Gab2 (p97) gene and methods of use thereof

ABSTRACT

This invention relates to the purification, cloning and characterization of a novel gene, Gab2. In response to extracellular stimuli (e.g., cyokines, growth factors, hormones and antigens), Gab2 binds several signal relay molecules, including the protein-tyrosine phosphatase SHP-2 and phosphatidylinositol-3-OH kinase (PI-3K), which results in the initiation of multiple signaling cascades. Gab2 nucleic acid molecules, peptides, vectors, host cells, probes, antibodies, knockout and transgenic animals are provided. The invention also relates to methods of diagnosis, prevention and treatment of Gab2-mediated conditions such as allergic responses, neoplastic disorders and immune disorders.

RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/US01/47854, which designated the United States and was filedOct. 26, 2001, published in English, and which claimed the benefit ofU.S. Provisional Application No. 60/243,495, filed Oct. 26, 2000. Theteachings of both Applications are incorporated herein by reference intheir entirety.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by a grants R01DK50693, P01 DK50654, R01 DK50654 and R01 CA49152 from the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] Extracellular stimuli are involved in a number of biologicalprocesses including cellular proliferation and differentiation. Severalcomponents in such biological processes (e.g., signaling cascades)remain unidentified. Thus, there is a need to identify new components ofsignaling cascades as well as to develop new, improved and effectivemethods to identify components of signaling cascades.

[0004] Allergies are a major medical problem with significant morbidityand even occasional mortality. Current anti-allergy drugs includeprimarily anti-histamines, cromolyl sodium, and steroids. Steroids canhave undesirable side effects. Cromolyn sodium is mainly useful forchronic prevention and often is an irritant in its own right.Antihistamines block the effects of mast cell degranulation, but do notprevent degranulation itself. There is a large market for newanti-allergy agents, as evinced by the success of non-sedatingantihistamines when they were introduced several years ago.

[0005] Cancer progression is a multi-step process, which evolves fromthe accumulation of mutations and the deregulation of the genes involvedin cell-growth control. In the case of human breast cancer, commonlyobserved genetic abnormalities include the loss of heterozygosity (LOH)in tumor suppressor genes and the DNA amplification and/oroverexpression of growth promoting oncogenes (e.g., c-myc, cyclin D1,and ErbB2/Neu). Breast carcinogenesis due to amplification of ErbB2 as aresult of Gab2 overexpression may provide not only a new diagnosticmarker for breast cancer detection but also an alternative therapeuticstrategy for treating some breast tumors.

[0006] Since Gab2 knockout mice are essentially healthy except fordefects in allergic response, intervention to specifically lower theexpression of Gab2 in vivo should have minimal side effects.

SUMMARY OF THE INVENTION

[0007] The invention relates to the isolation, cloning, sequencing andcharacterization of the Gab2 gene or a fragment, derivative or mutationthereof. The present invention also relates to DNA molecules (alsoreferred to herein as DNA sequences or nucleic acid sequences) whichencode a protein which comprises Gab2. The present invention alsorelates to DNA sequences capable of hybridizing to the DNA sequence ofGab2.

[0008] The present invention further relates to an expression vectorcomprising the nucleic acid molecule of Gab2, or a fragment, derivativeor mutation thereof. The present invention also relates to a host cellwhich has been transformed, or transfected, with the expression vectorcomprising the nucleic acid molecule of Gab2, or a fragment, derivativeor mutation thereof.

[0009] The protein, or peptides, of the present invention can be used toproduce antibodies, both polyclonal and monoclonal, which are reactivewith (i.e., bind to) the Gab2 protein, and can be used in diagnosticassays to determine the presence or type of a Gab2-mediated, e.g.,Gab2-dependent, disease.

[0010] The invention further relates to a transgenic non-human mammal(e.g., a mouse) with a disruption of the Gab2 gene in its genome, eithera homozygous disruption or a heterozygous disruption, such that themammal lacks, or has reduced levels of, functional Gab2 protein. Theinvention also relates to a transgenic non-human mammal (e.g., a mouse)in which the genome has been altered such that the mammal has increasedlevels of functional Gab2 protein. These transgenic mammals wouldexhibit an altered responsiveness to cytokine, growth factor, hormone orantigen stimulation.

[0011] The invention further relates to the use of an agent whichinhibits a Gab2 interaction with an associated protein, in response toan extracellular stimulus (e.g., a cytokine, growth factor, hormone orantigen) for the manufacture of a medicament for preventing or treatinga GAb2-mediated injury (e.g., an allergic response, a neoplasticdisease, or an immune disorder).

[0012] The agent of the present invention can be selected from the groupconsisting of proteins, polypeptides, antibodies, oligonucleotides,small molecules, natural product inhibitors, mutants of Gab2, andmutants of Gab2-associated molecules, or wherein the agent is anoligonucleotide antisense to the nucleic acid sequence of Gab2, orantisense to a Gab2 homolog, fragment, complementary sequence, ormutant.

[0013] The invention further relates to the nasal, topical or systemicadministration of the agent in which the agent is a mutant Gab2, orfragment thereof, which competes with Gab2 for interaction with itsassociated proteins, or the agent inhibits the expression of Gab2 or theagent inhibits the tyrosyl phosphorylation of Gab2. The invention alsorelates to the administration of the agent as an insert in a genetherapy vector.

[0014] The agent of the present invention can also be employed toinhibit the response of mast cells to FceRI receptor stimulation byadministration of the agent to the mast cells. In particular, the agentcan prevent a Gab2-mediated injury, for example, an allergic response,by inhibiting a Gab2 interaction with an associated protein in responseto an extracellular signal and, thus, prevent activation of aGab2-mediated signaling cascade.

[0015] The agent of the present invention can also be employed toinhibit a neoplastic disease (e.g., leukemia, prostate cancer and breastcancer) by inhibiting a Gab2 interaction with an associated protein inresponse to an extracellular signal and, thus, prevent activation of aGab2-mediated signaling cascade. The Gab2 nucleic acid sequence of thepresent invention can be used to produce a probe that can detectupregulation of Gab2 product in a patient with a neoplastic disorder.

[0016] The invention also relates to a method of identifying a drug tothat can be administered to treat a Gab2-mediated condition by producinga mouse that is a model of the condition, and administering to the mousea drug to be assessed for its effectiveness in treating or preventingthe condition. If the drug reduces the extent to which the condition ispresent or progresses, the drug is a drug to be administered to treatthe condition.

[0017] The present invention also relates to isolated RNA molecules(double-stranded; single-stranded) which mediate RNAi of Gab2. That is,the isolated RNAs of the present invention mediate degradation of Gab2mRNA. It is not necessary that there be perfect correspondence of thesequences, but the correspondence must be sufficient to enable the RNAto direct RNAi cleavage of the Gab2 mRNA. In a particular embodiment,the RNA molecules of the present invention comprise a 3′ hydroxyl group.

[0018] The present invention also relates to RNA produced by the methodsof the present invention, as well as to RNAs, produced by other methods,such as chemical synthesis or recombinant DNA techniques, that have thesame or substantially the same sequences as naturally-occurring RNAsthat mediate Gab2 RNAi, such as those produced by the methods of thepresent invention. The invention further relates to uses of the RNAs,such as for therapeutic or prophylactic treatment and compositionscomprising RNAs that mediate Gab2 RNAi, such as pharmaceuticalcompositions comprising RNAs and an appropriate carrier (e.g., a bufferor water).

[0019] The present invention also relates to a method of mediating RNAinterference of Gab2 mRNA of a gene in a cell or organism (e.g., mammalsuch as a mouse or a human). In one embodiment, RNA which targets theGab2 mRNA is introduced into the cell or organism. The cell or organismis maintained under conditions under which degradation of the mRNAoccurs, thereby mediating RNA interference of the Gab2 mRNA in the cellor organism. As used herein, the term “cell or organism in which RNAioccurs” includes both a cell or organism in which Gab2 RNAi occurs asthe cell or organism is obtained, or a cell or organism that has beenmodified so that RNAi occurs.

[0020] The present invention also relates to a method for knocking down(partially or completely) the Gab2 gene, thus providing an alternativeto presently available methods of knocking down (or out) the Gab2 gene.This method of knocking down gene expression can be used therapeuticallyor for research (e.g., to generate models of disease states, to examinethe function of a gene, to assess whether an agent acts on a gene, tovalidate targets for drug discovery). In those instances in which genefunction is eliminated, the resulting cell or organism can also bereferred to as a knockout. One embodiment of the method of producingknockdown cells and organisms comprises introducing into a cell ororganism in which Gab2 is to be knocked down, RNA that targets the Gab2gene and maintaining the resulting cell or organism under conditionsunder which RNAi occurs, resulting in degradation of the Gab2 mRNA,thereby producing knockdown cells or organisms. Knockdown cells andorganisms produced by the present method are also the subject of thisinvention.

[0021] The present invention also encompasses a method of treating adisease or condition associated with the presence of the Gab2 protein inan individual comprising administering to the individual RNA whichtargets the Gab2 mRNA for degradation. As a result, the protein is notproduced or is not produced to the extent it would be in the absence ofthe treatment.

[0022] Also encompassed by the present invention is a method ofidentifying target sites within a Gab2 mRNA that are particularlysuitable for RNAi as well as a method of assessing the ability of RNAsto mediate RNAi of Gab2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a schematic of the HPLC of Lys-C fragments from Gab2.Nine peaks (numbered) were selected for Edman sequencing.

[0024]FIG. 2 depicts the predicted protein sequence derived from Gab2cDNA (SEQ ID NO: 5). The locations of 8 of the 9 peptides obtained byEdman sequencing are underlined. PH domain is in bold,tyrosine-containing motifs are indicated (- -), and PXXP motifs are inbold.

[0025]FIG. 3 is a schematic illustration comparing Dos, Gab2 (p97) andGab1. Percentage sequence identity among family members is indicated.(P)=potential proline-rich domains.

[0026]FIG. 4 depicts the alignment of Gab/Dos family PH domains (top)and the MBD region in Gab1 and Gab2 (p97)(bottom). β1 and β2 sheetscorresponding to the PH domain of PLCδ1 are indicated. Basic amino acidresidues between β1 and β2 are in bold. A proposed consensus that mayspecify Gab/Dos family members is indicated. Two PXXP motifs within Gab1are overlined.

[0027]FIG. 5A-FIG. 5E depict the effects of the SHP-2/Gab2 complex onIL-3-induced transactivation, and IL-3-induced MAPK activation. (A)Effects of expression of Gab2ΔY2HA on IL-3-induced c-fos promoteractivity. (B) Effects of Gab2/ΔY2HA on IL-3 induced c-fos promoteractivity. (C) Effects of point mutations in Gab2 on IL-3-induced c-fospromoter activity. (D) Normalized GAL-4 luciferase activity of BaF3cells transiently co-transfected with the indicated expression vectorsor vector alone, together with a GAL4-Elk-1 construct and GAL-4- andTK-Renilla luciferase reporters. (E) Effects of Gab2/SHP-2 onSTAT-mediated activation.

[0028]FIG. 6 depicts the nucleotide sequence of Gab2 (SEQ ID NO: 6).

[0029]FIG. 7 depicts the targeting strategy for the generation of Gab2knockout mice.

[0030]FIG. 8 depicts surface expression of Fcε RI in Gab2−/− BMMCs.

[0031]FIG. 9 depicts Fcε RI-mediated degranulation in Gab2−/− BMMCs.

[0032]FIG. 10 depicts Fcε RI-evoked TNFα and IL-6 gene expression inGab2−/− BMMCs.

[0033]FIG. 11 depicts passive cutaneous anaphylaxis in wild-type (WT)and Gab2−/− mice.

[0034]FIG. 12 depicts a schematic illustration of the Gab2 response toFcε RI receptor stimulation.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Gab2, a novel member of the DOS/Gab1 subfamily of scaffoldingmolecules, was purified, cloned and characterized. DOS/Gab1 Familymembers contain an N-terminal PH (Pleckstrin homology) domain,proline-rich motifs and multiple tyrosines, but show minimal sequenceidentity, mainly in their PH domains (FIGS. 2, 4) (see Rameh, L. E. etal., J. Biol. Chem. 272, 22059-22066 (1997)). Most of their tyrosylresidues, including those required for SHP-2 binding (Table 2 and FIG.3), occur at similar relative positions and within similar sequencecontexts (FIG. 3). This suggests, that Gab/Dos proteins bind similarsignal relay molecules, which may have to bind in a specificorientation. IRS proteins and FRS-2 have similar topography, but aredistinguished from Dos/Gab proteins by their PH domains and the orderand sequence contexts of their tyrosines and proline-rich motifs.Members of the family include Drosophila Dos and mammalian Gab1.Tissue-specific differences in Gab1 and Gab2 expression exist, and atleast one functionally important region of Gab1, the MBD, is divergentin Gab2. Although Gab2 and Gab1 are only distantly related and havedistinct functions in vivo, the novel protein is termed Gab2 to simplifythe nomenclature. Gab2 is also known as p97 and p97/Gab2.

[0036] Extracellular stimuli, such as cytokines, growth factors,hormones and antigens, regulate cell proliferation and differentiationvia changing the tyrosine phosphorylation states of proteins, such asGab2, inside the cell. Extracellular receptors, such as cytokinereceptors, trigger multiple signaling cascades, which regulate cellproliferation and differentiation. Binding of a cytokine to its cognatereceptor activates receptor-associated Janus family (e.g., Jak/Tyk)protein-tyrosine kinase(s) (PTKs), resulting in their phosphorylationand the phosphorylation of receptor cytoplasmic domains. Phosphorylationcreates docking sites for SH2 domain-containing signal relay molecules,most of which are substrates for Janus PTKs. Signal relay moleculespromote activation of downstream cascades, including theRas/Raf/mitogen-activated protein Kinase (MAPK), phosphatidylinositol-3′kinase (PI-3K) and Stat cascades. Ultimately, these cascades evoke thetranscription of immediate-early genes, such as c-fos (see Ihle, J. N.et al., Stem Cells 15, 105-111 (1997)). Receptor tyrosine kinases (RTKs)and multi-chain immune recognition receptors (MIRRs) utilize analogoussignaling strategies.

[0037] Gab2 is widely expressed in a variety of tissues, and containsmultiple potential serine/threonine phosphorylation sites. Uponcytokine, growth factor, hormone or antigen receptor stimulation, Gab2becomes tyrosine phosphorylated, most likely by the associated JanusPTK, and involved in the activation of multiple signaling cascades viainteraction with different intracellular signaling molecules. Upontyrosine phosphorylation, Gab2 recruits SH2-containing downstream signalmolecules. Gab2 may also transmit extracellular stimuli throughinteraction with cellular proteins containing SH3 or WW domains. Inaddition, Gab2 may exert its effect through an interaction withintracellular lipids.

[0038] In various hematopoietic cell lines, in response to cytokinestimulation and engagement of MIRRs, Gab2 becomes associated withvarious SH2-containing molecules, including SHP-2, p85 (the regulatorysubunit of PI-3K) and Shc. Gab2, via its interaction with SHP-2, isrequired for cytokine (e.g., IL-3) induced c-fos gene transcription viaa novel mechanism that is parallel to or downstream of MAPK FIGS. 5A-E).However, Gab2 can also signal to the MAPK cascade under some conditions.This signal does not require SHP-2 binding since Gab2 mutants which donot bind SHP-2 potentiate (do not inhibit) MAPK activation. Conceivably,Gab2 may transmit signals to the MAPK cascade via PI-3K.

[0039] In several systems, PI-3K functions downstream of Ras andupstream of MAPK, particularly under conditions of limiting receptorstimulation. The Gab2/p85 complex is critical for PI-3K activationevoked by cytokine receptors, such as IL-3/GM-CSF and IL-2, which do notcontain p85-binding sites. However, Gab2 is also tyrosyl phosphorylatedupon stimulation of receptors that bind p85 directly (e.g., CSF-1R) orhave other means of p85 recruitment (e.g., CD19 for the BCR). In thesesystems, Gab2 may amplify PI-3K activation.

[0040] Upon cytokine stimulation, Gab2 can be recruited to receptorcomplexes. PH domains bind to phosphatidylinositol lipids, providing apotential recruitment mechanism. Alternatively, the Gab2 regioncorresponding to the Gab1 MBD may direct binding. A third possibility isthat recruitment is indirect. Mutation of Y577 in the IL-3 receptor pcchain, the Shc binding site, severely diminishes Gab2 tyrosylphosphorylation (see Itoh, T. et al., J. Biol. Chem. 271, 7587-7592(1996)). Since Grb2 (presumably via its SH3 domains) binds Gab2constitutively, and the Grb2 SH2 domain binds to tyrosyl phosphorylatedShc, Gab2 recruitment to the IL-3 receptor may occur via a Shc-Grb2complex.

[0041] Role of Gab2 in Allergic Responses

[0042] Interaction of multivalent antigen with IgE-bound mast cellsprovokes several effects: the immediate release of preformed granulescontaining vasoactive amines including histamine and serotonin(degranulation), secretion of lipid mediators, and the late synthesisand release of cytokines. Fcε RI is the high affinity receptor for IgE.Fcε RI consists of a ligand binding α chain, one β, and two γ chains.Crosslinking Fcε RI by the multivalent antigen activates the P chainassociated with tyrosine kinase, lyn. Activated lyn phosphorylates thetyrosine-based activation motifs (IAMTs) in the cytoplasmic domains ofthe P and y chains, which recruit and activate tyrosine kinase Syk.Subsequently, lyn and syk phosphorylate various signal relay molecules,resulting in the activation of multiple downstream signaling cascadesincluding phosphatidylinositol 3-kinase (PI-3K) and three majorsubfamilies of mitogen-activated protein kinases (MAPKs), Erk, JNK, andp38. The lipid products of PI-3K are required for full activation of Tecfamily tyrosine kinase Btk/Emt, and subsequent phosphorylation andactivation of PLCy. Activated PLCy then converts P14,5P2 into inosital1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which can increaseintracellular Ca⁺⁺ level and activate PKC respectively. Both Ca⁺⁺ andPKC are required for the optimal degranulation. Activation of Erk, JNK,and p38 are important for the late phase cytokine production.

[0043] Since one of the major functions of mast cells is involved inIgE-associated immune responses, the role of Gab2 in signaling evoked byFcε RI in Bone Marrow-derived Mast Cells (BMMCS) was determined. Gab2knockout mice were generated by conventional gene targeting methods(FIG. 7). Bone marrow mast cells or macrophages were isolated from thesemice by standard techniques. To examine Fcε signaling, mast cells wereloaded with anti-DNP IgE and then stimulated with a range ofconcentrations of DNP, and serotonin release was quantified. Forassessment of macrophage Fcy responses, bone marrow macrophages werepresented with opsonized RBCs, and phagocytosis was quantified bymicroscopy. In addition, various biochemical parameters were monitoredby standard techniques.

[0044] In contrast to Gab1 knockout mice, which are embryonic lethal,Gab2 homozygous (−/−) knockout mice are healthy, fertile and appear tolive a normal life span. However, analysis of mast cells from Gab2−/−mice show that they are refractory to stimulation through IgE receptorswhich are the main receptors mediating allergic responses. Mast cellnumbers are also diminished in Gab2−/− mice, and Fe gamma receptorsignaling in macrophages is defective. Thus, elimination of Gab2expression, or Gab2 interaction with key signaling molecules, is apowerful approach to prevention of allergic responses.

[0045] The mast cell degranulation evoked by Fcε RI crosslinking wasdramatically impaired (e.g., a 3-7 fold impairment of Fcε-evokedserotonin release) in Gab2−/− BMMCS compared to wild type BMMCS (FIG.9). Fcε RI-evoked PLCγ phosphorylation and interleukin-3-induced AKTactivation also are significantly diminished in Gab2−/− BMMCS, as wellas Fcε RI evoked TNFα and IL-6 gene expression (FIG. 10). Furthermore,activation of JNK and p38 are defective in Gab2−/− upon Fcε RIcrosslinking. In addition, Gab2−/− mice show decreased passive cutaneousanaphylaxis compared to wild mice (FIG. 11). These data show that Gab2is required for selective events downstream of Fcε activation, mostlikely those dependent on PI-3K activation.

[0046] These defects in Fcε RI-evoked biological response and signalingcascades in Gab2−/− BMMCS are not due to a change in surface expressionof Fε RI (FIG. 8) or defective activation of upstream tyrosine kinases.Notably, early events in Fee responses, including surface expression ofthe IgE receptor, Syk phosphorylation and, most likely, LATphosphorylation, as well as Erk MAPK activation, are normal.Furthermore, total tyrosyl phosphorylation in Gab2−/− BMMCs is normalupon FcεRI engagement. Moreover, expression of the wild type Gab2 inGab2−/− BMMCS can rescue the FcεRI-evoked signaling defects. Thus, thesedata demonstrate that Gab2 plays an important role in FcεRI-evokedsignaling and effector function in mast cells and is essential for theIgE-initiated effector function of mast cells by activating thePI-3K/Akt and JNK and p38 cascades (FIG. 12). Similar results areobtained in macrophages, where Fcγ-evoked phagocytosis is impaired by upto 50%. Gab2 and its associated signaling molecules represent newtargets for developing drugs to treat allergy.

[0047] Role of Gab2 Interaction with SHP-2

[0048] In another embodiment, the Gab2/SHP-2 complex also has a distinctand novel signaling role, since Gab2 mutants inhibit activation of c-fosluciferase (FIG. 5A) and Elk (i.e, TCF)-(FIG. 5D) and STAT-(FIG. 5E)driven reporters. The N-SH2 of SHP-2 binds to tyrosyl phosphorylatedGab2, and N-SH2 engagement activates the enzyme (see Barford, D. andNeel, B. G., Structure 6, 249-254 (1998)). Dominant negative SHP-2blocks Gab2 potentiation of basal c-fos promoter activity (FIG. 5A),suggesting that upon binding to Gab2, activated SHP-2 dephosphorylatesone or more targets to permit c-fos activation. The identity of thistarget(s) remains unknown. Previous experiments with “substratetrapping” mutants suggested that Gab2 is a SHP-2 substrate, and Dosreportedly is a substrate of Csw. However, mutating its SHP-2 bindingsites does not increase Gab2 tyrosyl phosphorylation. Conceivably, theincreased association of the cystein to serine mutant (C>S) of SHP-2with tyrosyl phosphorylated Gab2 is due to SHP-2 regulation of a PTKthat phosphorylates Gab2, with consequently increased SHP-2 binding viaits SH2 domains to Gab2. However, it remains possible that one or moresites on Gab2 are SHP-2 targets. SHP-2 could dephosphorylate its ownbinding sites. Alternatively, it could target other sites, but the netincrease in their phosphorylation might be less than the decreasedphosphorylation due to loss of the SHP-2 sites. If sites on Gab2 are notthe primary target(s) of the Gab2/SHP-2 complex, then presumably Gab2directs SHP-2 to the proper location for accessing its target(s).

[0049] Although Gab2 is strongly tyrosyl phosphorylated in response tomany stimuli, its association with SHP-2 varies, suggesting that Gab2may be phosphorylated on distinct sites in response to differentstimuli. The widespread expression of Gab2 suggests that its involvementin growth factor and/or cytokine signaling cascades in non-hematopoieticcells should be investigated.

[0050] SHP-2 is a critical component of multiple signaling cascades.Embryonic stem cells expressing mutant SHP-2 exhibit defective ex vivohematopoietic differentiation. SHP-2 is required for cytokine-inducedMAPK activation. For example, SHP-2 is required for interleukin-5(IL-5)-induced MAPK activation in eosinophils, interleukin-2(IL-2)-induced MAPK activation in T cells, and for immediate-early geneactivation in response to multiple cytokines in various cellularcontexts. RTK and T cell receptor (TCR)-evoked MAPK activation alsorequire SHP-2, and SHP-2 function is necessary for RTK-induced c-fosexpression acting, at least in part, to control the activity of thetranscription factor Elk-1. Likewise, the Drosophila homolog of SHP-2,Csw, is required for RTK-induced gene induction. These studies suggestthat SHP-2 is a required positive component of cytokine, RTK, and MIRRsignaling cascades, acting upstream of MAPK, which, in turn, liesupstream of immediate-early genes.

[0051] However, several lines of evidence raise questions about thissimple linear model. For example, SHP-2 has been reported to act bothupstream and downstream of Ras. Genetic analysis indicates that Csw actsupstream and downstream of Ras or functions in a parallel cascade inSevenless signaling. Biochemical and genetic studies suggest that Cswbinds to, dephosphorylates, and signals through the Daughter ofSevenless (Dos) gene product, a scaffolding protein remotely related tomammalian Gab1. SHP-2 binds directly to some growth factor receptors,but in other cascades, it binds to scaffolding molecules such as IRSfamily members, Gab 1, and FRS-2, and/or to one or more transmembraneglycoproteins, such as SHPS-1/SIRPs.

[0052] Accordingly, upon cytokine, growth factor, hormone or antigenreceptor (B cell receptor/T cell receptor) stimulation, Gab2 becomestyrosyl phosphorylated and associates with several SH2 domain-containingproteins, including SHP-2. Gab2 via interaction with SHP-2 is requiredfor cytokine induced gene expression, via a novel signaling cascade.Expressed Gab2 mutants that were unable to bind SHP-2 blockscytokine-induced c-fos promoter activation, inhibiting Elk-1-mediatedand STAT5-mediated transactivation, indicating that Gab2 function isrequired for cytokine-induced immediate early gene activation. Inaddition, dominant negative SHP-2 inhibits IL-3-induced MAPK activationin BaF3 cells. However, SHP-2 need not bind to Gab2 to participate inMAPK activation, whereas it must bind to Gab2 to allow transcriptionalactivation (FIGS. 5A-E). Thus, SHP-2 has at least two sites of action incytokine signaling. The requirement of SHP-2 for MAPK activation couldbe mediated through another binding protein, e.g, p135 in BaF3 cells(see Gu, H. et al., J. Biol. Chem. 272, 16421-16430 (1997)), or throughthe reported direct interaction of SHP-2 and Jak-2 (see Fuhrer, D. K. etal., J. Biol. Chem. 270, 24826-24830 (1995); Ali, S. et al., EMBO J. 15,135-142 (1996)), whereas the work herein shows that the requirement ofSHP-2 for transactivation is mediated by Gab2. SHP-2 action at multiplesteps may help explain some of the discrepancies between earlier studiesover the site of action of SHP-2 in RTK signaling. Future experimentscan be directed to elucidating Gab-2-dependent and -independentfunctions of SHP-2 in signaling by cytokines, growth factors, and MIRRs.How these interactions culminate in MAPK activation and/or induction ofgene expression is unclear. To understand how SHP-2 functions requiresboth the identification and characterization of its binding proteins andsubstrates.

[0053] Gab2 is an important new regulator of receptor signaling thatcontrols a novel cascade to immediate-early gene activation and suggestnew functions for SHP-2 in cytokine receptor signaling. Moreover, sinceSHP-2 itself is required for IL-3-induced MAPK activation, these datashow that SHP-2 is required for at least two steps in cytokine signaltransduction, one upstream of, and the other downstream of or parallelto MAPK.

[0054] Gab2/SHP-2 may be required for MAPK translocation because MAPKsprobably must translocate to the nucleus to phosphorylate transcriptionfactors (e.g., Elk-1) (see Brunet, A. and Pouyssegur, J., Essays Biochem32, 1-16 (1997)). Consistent with this model, MAPK activation also isrequired for IL-3-driven STAT reporter activity in BaF3 cells (seeRajotte, D. et al., Blood 88, 2906-2916 (1996)).

[0055] Gab2/SHP2 may control a cascade that inhibits dephosphorylationof SRF, TCF, and/or other components of the c-fos transcriptionalmachinery, perhaps by controlling the serine-threonine kinase KSR.Recent work suggests that KSR activates a serine-threonine phosphatasethat catalyzes Elk-1 dephosphorylation. Interestingly, IRS-1 alsosignals to c-fos without affecting MAPK. Data suggest that this signalmay be sent via IRS-1/SHP-2 complexes; by inference, similar signalingcascades may exist for other scaffolding protein/SHP-2 complexes.Whereas Gab2/SHP-2 complex formation appears to be required for fullactivity of the c-fos promoter, Gab2 mutants unable to bind SHP-2 onlypartially inhibit c-fos activation. It is unclear whether partialinhibition is due to incomplete interference with endogenous Gab2 by theGab2 mutants or instead indicates that Gab2/SHP-2 is predominantly an“amplifier” of c-fos activation. The latter is likely because theeffects of the Gab2 mutants on c-fos promoter activity are enhanced atlower levels of IL-3. Moreover, the results described herein do notexclude important roles for Gab2 and/or SHP-2 in cascades other thanthose leading to c-fos promoter activation.

[0056] Role of Gab2 in Neoplastic Disorders

[0057] Overexpression of Gab2 may also play a role in neoplasticdisorders. It may promote breast carcinogenesis, for example, byamplifying EGFR/ErbB2 initiated growth and survival signals. ErbB2/Neuis the best known gene that is amplified and overexpressed in humanbreast cancer. Although oncogenic mutant forms of ErbB2 are rarely foundin humans, ErbB2 amplification and overexpression has been identified in20-30% of all the breast cancer cases and is correlated with poorpatient prognosis. Interestingly, ErbB2/Neu overexpression is found in˜90% of Ductal Carcinoma in situ (DCIS), a malignant ductal carcinomawith an intact basement membrane barrier. Under in vitro cultureconditions, EGFR(ErbB1) signals have been shown to contribute todisorganized growth of colonies of breast tumor cells under athree-dimensional basement membrane, which mimics DCIS. Furthermore,overexpression of ErbB2 under the control of mouse mammary tumor virus(MMTV) long terminal repeat (LTR) in mammary gland causes mammary tumorformation with fairly long latency in mice. Consistent with a role ofErbB2 in breast cancer, ErbB2 blocking antibodies have been usedclinically to reduce regression of some of the ErbB2-overexpressingbreast tumors. Collectively, these data indicate that ErbB2/neuoverexpression contributes to breast carcinogenesis in human.

[0058] ErbB2 belongs to the EGFR/ErbB receptor tyrosine kinase family,which also includes ErbB1/EGFR, ErbB3, and ErbB4. ErbB members can formhomodimers or heterodimers depending on binding to specific ligand.Although no ligand for ErbB2 has been identified, ErbB2 acts as aco-receptor for ErbB 1, ErbB3, and ErbB4 when the latter binds to EGFfamily members or neuregulin respectively. Upon ligand binding, ErbBmembers become dimerized and activated. Activated ErbBs phosphorylatevarious tyrosine residues in the cytoplasmic domain, resulting in therecruitment and activation of various downstream signaling cascadesincluding ras/raf/MAPK, phosphatidylinositol-3 kinase (PI-3K), andtyrosine phosphatase SHP-2, which provide signals for various cellularresponses including proliferation and survival. It is still not clearhow each of the ErbB-activated cascades contribute to breastcarcinogenesis in vivo. Nevertheless, since ErbB2 overexpressioninducing breast tumor formation suggests that enhanced PTK activity orErbB downstream signal relay/adapter components may contribute to breastcarcinogenesis.

[0059] Although Gab-like molecules have not been implicated in breastcancer previously, overexpression of Gab1 in fibroblasts has been shownto potentiate EGF-mediated cell growth and transformation and anchorageindependent growth, and Gab2 is important for cytokine-induced cellgrowth via its ability to activate PI-3K. Using fluorescence in situhybridization (FISH) analysis, the Gab2 gene was located on humanchromosome 11q13.3-14.2. Since chromosome 11q13 amplification has beenfound in 10-15% of breast cancer patients, Gab2 expression in breastcancer cell lines and tumors was examined and Gab2 was found to beoverexpressed in ˜40% of breast cancer cell lines and 20% of primarybreast tumor samples tested. Although Gab2 tyrosine phosphorylation andits associated PI-3K activity have been correlated with EGF-induced cellproliferation, the functional evolvement of Gab2 in EGF-mediated cellgrowth has not been fully investigated.

[0060] In one embodiment, the invention encompasses Gab2, its homologs,analogs, variants, mutants, complementary nucleic acid sequences.Encompassed by the present invention are proteins that havesubstantially the same amino acid sequence as Gab2, or polynucleotidesthat have substantially the same nucleic acid sequence as thepolynucleotides encoding Gab2. “Substantially the same sequence” means anucleic acid or polypeptide that exhibits at least about 90% sequenceidentity with a reference sequence, e.g., another nucleic acid orpolypeptide, preferably at least about 95% identity, and more preferablyat least about 97% sequence identity with the reference sequence. Thelength of comparison for sequences will generally be at least 75nucleotide bases or 25 amino acids, more preferably at least 150nucleotide bases or 50 amino acids, and most preferably 243-264nucleotide bases or 81-88 amino acids. “Polypeptide” as used hereinindicates a molecular chain of amino acids and does not refer to aspecific length of the product. Thus, peptides, oligopeptides andproteins are included within the definition of polypeptide. This term isalso intended to include polypeptide that have been subjected topost-expression modifications such as, for example, glycosylations,acetylations, phosphorylations and the like.

[0061] “Sequence identity,” as used herein, refers to the subunitsequence similarity between two polymeric molecules, e.g., twopolynucleotides or two polypeptides. When a subunit position in both ofthe two molecules is occupied by the same monomeric subunit, e.g., if aposition in each of two peptides is occupied by serine, then they areidentical at that position. The identity between two sequences is adirect function of the number of matching or identical positions, e.g.,if half (e.g., 5 positions in a polymer 10 subunits in length) of thepositions in two peptide or compound sequences are identical, then thetwo sequences are 50% identical; if 90% of the positions, e.g., 9 of 10are matched, the two sequences share 90% sequence identity. By way ofexample, the amino acid sequences R₂R₅R₇R₁₀R₆R₃ and R₉R₈R₁R₁₀R₆R₃ have 3of 6 positions in common, and therefore share 50% sequence identity,while the sequences R₂R₅R₇R₁₀R₆R₃ and R₈R₁R₁₀R₆R₃ have 3 of 5 positionsin common, and therefore share 60% sequence identity. The identitybetween two sequences is a direct function of the number of matching oridentical positions. Thus, if a portion of the reference sequence isdeleted in a particular peptide, that deleted section is not counted forpurposes of calculating sequence identity, e.g., R₂R₅R₇R₁₀R₆R₃ andR₂R₅R₇R₁₀R₃ have 5 out of 6 positions in common, and therefore share83.3% sequence identity.

[0062] Identity is often measured using sequence analysis software e.g.,BLASTN or BLASTP (available at http://www.ncbi.nlm.nih.gov/BLAST/). Thedefault parameters for comparing two sequences (e.g., “Blast”-ing twosequences against each other, http://www.ncbi.nlm.nih.gov/gorf/bl2.html)by BLASTN (for nucleotide sequences) are reward for match=1, penalty formismatch=−2, open gap=5, extension gap=2.

[0063] When using BLASTP for protein sequences, the default parametersare reward for match=0, penalty for mismatch=0, open gap=11, andextension gap=1.

[0064] When two sequences share “sequence homology,” it is meant thatthe two sequences differ from each other only by conservativesubstitutions. For polypeptide sequences, such conservativesubstitutions consist of substitution of one amino acid at a givenposition in the sequence for another amino acid of the same class (e.g.,amino acids that share characteristics of hydrophobicity, charge, pK orother conformational or chemical properties, e.g., valine for leucine,arginine for lysine), or by one or more non-conservative amino acidsubstitutions, deletions, or insertions, located at positions of thesequence that do not alter the conformation or folding of thepolypeptide to the extent that the biological activity of thepolypeptide is destroyed. Examples of “conservative substitutions”include substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another; the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between threonine andserine; the substitution of one basic residue such as lysine, arginineor histidine for another; or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another; or the use of achemically derivatized residue in place of a non-derivatized residue;provided that the polypeptide displays the requisite biologicalactivity. Two sequences which share sequence homology may called“sequence homologs.”

[0065] Homology, for polypeptides, is typically measured using sequenceanalysis software (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Protein analysis softwarematches similar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; senne, threonine; lysine,arginine; and phenylalanine, tyrosine.

[0066] The invention also contemplates mutants of the proteins andpeptides disclosed herein, where the mutation(s) do not substantiallyalter the activity of the protein or peptide, that is the mutations areeffectively “silent” mutations.

[0067] By “mutant” of Gab2 is meant a polypeptide that includes anychange in the amino acid sequence relative to the amino acid sequence ofthe equivalent reference Gab2 polypeptide. Such changes can arise eitherspontaneously or by manipulations by man, by chemical energy (e.g.,X-ray), or by other forms of chemical mutagenesis, or by geneticengineering, or as a result of mating or other forms of exchange ofgenetic information. Mutations include, e.g., base changes, deletions,insertions, inversions, translocations, or duplications. Mutant forms ofGab2 may display either increased or decreased activity relative to theequivalent reference Gab2 polynucleotide, and such mutants may or maynot also comprise additional amino acids derived from the process ofcloning, e.g., amino acid residues or amino acid sequences correspondingto full or partial linker sequences.

[0068] Mutants/fragments of the protein of the present invention can begenerated by PCR cloning. To make such fragments, PCR primers aredesigned from known sequence in such a way that each set of primers willamplify known subsequence from the overall protein. These subsequencesare then cloned into an appropriate expression vector and the expressedprotein tested for its activity as described in the assays describedherein.

[0069] Mutants/fragments of the protein of the present invention canalso be generated by Pseudomonas elastase digestion, as described byMariyama, M. et al. (1992, J. Biol. Chem. 267:1253-8).

[0070] By “analog” of Gab2 is meant a non-natural molecule substantiallysimilar to either the entire Gab2 molecule or a fragment or allelicvariant thereof, and having substantially the same or superiorbiological activity. Such analogs are intended to include derivatives(e.g., chemical derivatives, as defined above) of the biologicallyactive Gab2, as well as its fragments, mutants, homologs, and allelicvariants, which derivatives exhibit a qualitatively similar agonist orantagonist effect to that of the unmodified Gab2 polypeptide, fragment,mutant, homolog, or allelic variant.

[0071] By “allele” of Gab2 is meant a polypeptide sequence containing anaturally-occurring sequence variation relative to the polypeptidesequence of the reference Gab2 polypeptide. By “allele” of apolynucleotide encoding the Gab2 polypeptide is meant a polynucleotidecontaining a sequence variation relative to the reference polynucleotidesequence encoding the reference Gab2 polypeptide, where the allele ofthe polynucleotide encoding the Gab2 polypeptide encodes an allelic formof the Gab2 polypeptide.

[0072] It is possible that a given polypeptide may be either a fragment,a mutant, an analog, or alielic variant of Gab2, or it may be two ormore of those things, e.g., a polypeptide may be both an analog and amutant of the Gab2 polypeptide. For example, a shortened version of theGab2 molecule (e.g., a fragment of Gab2) may be created in thelaboratory. If that fragment is then mutated through means known in theart, a molecule is created that is both a fragment and a mutant of Gab2.In another example, a mutant may be created, which is later discoveredto exist as an allelic form of Gab2 in some mammalian individuals. Sucha mutant Gab2 molecule would therefore be both a mutant and an allelicvariant. Such combinations of fragments, mutants, allelic variants, andanalogs are intended to be encompassed in the present invention.

[0073] Also encompassed by the present invention are chemicalderivatives of Gab2. “Chemical derivative” refers to a subjectpolypeptide having one or more residues chemically derivatized byreaction of a functional side group. Such derivatized residues includefor example, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups may be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-imbenzylhistidine. Also included as chemical derivatives arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids. For examples:4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstitute for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and omithine may besubstituted for lysine.

[0074] The present invention also includes fusion proteins and chimericproteins comprising the Gab2 protein, its fragments, mutants, homologs,analogs, and allelic variants. A fusion or chimeric protein can beproduced as a result of recombinant expression and the cloning process,e.g., the protein may be produced comprising additional amino acids oramino acid sequences corresponding to full or partial linker sequences,comprises additional vector sequence added to the protein, including aHA tag. As used herein, the term “fusion or chimeric protein” isintended to encompass changes of this type to the original proteinsequence. A fusion or chimeric protein can consist of a multimer of asingle protein, e.g., repeats of the Gab2 protein, or the fusion andchimeric proteins can be made up of several proteins. The term “fusionprotein” or “chimeric protein” as used herein can also encompassadditional components for e.g., delivering a chemotherapeutic agent,wherein a polynucleotide encoding the chemotherapeutic agent is linkedto the polynucleotide encoding the Gab2 protein.

[0075] Multimeric proteins comprising Gab2, its fragments, mutants,homologs, analogs and allelic variants are also intended to beencompassed by the present invention. By “multimer” is meant a proteincomprising two or more copies of a subunit protein. The subunit proteinmay be the protein of the present invention, e.g., Gab2 repeated two ormore times, or a fragment, mutant, homolog, analog or allelic variant,e.g., a Gab2 mutant or fragment, repeated two or more times. Such amultimer may also be a fusion or chimeric protein, e.g., a repeated Gab2mutant may be combined with polylinker sequence, and/or one or morepeptides, e.g., Gab2-associated peptides, which may be present in asingle copy, or may also be tandemly repeated, e.g., a protein maycomprise two or more multimers within the overall protein.

[0076] In one embodiment, the invention encompasses Gab2, its homologs,analogs, variants, mutants, complementary nucleic acid sequences, andsequences which can be used to design probes which hybridize to Gab2 orits complementary strand. The design of the probes should preferablyfollow these parameters: (a) it should be designed to an area of thesequence which has the fewest ambiguous bases (“N's”), if any, and (b)it should be designed to have a T_(m) of approx. 80° C. (assuming 2° C.for each A or T and 4 degrees for each G or C).

[0077] The probes should preferably be labeled with g-³²P-ATP (specificactivity 6000 Ci/mmole) and T4 polynucleotide kinase using commonlyemployed techniques for labeling oligonucleotides. Other labelingtechniques can also be used. Unincorporated label should preferably beremoved by gel filtration chromatography or other established methods.The amount of radioactivity incorporated into the probe should bequantitated by measurement in a scintillation counter. In oneembodiment, specific activity of the resulting probe should beapproximately 4×10⁶ dpm/pmole. The bacterial culture containing the poolof full-length clones can be thawed and 100 μl of the stock used toinoculate a sterile culture flask containing 25 ml of sterile L-brothcontaining ampicillin at 100 μg/ml. The culture can be grown tosaturation at 37° C., and the saturated culture should preferably bediluted in fresh L-broth. Aliquots of these dilutions can be plated todetermine the dilution and volume which will yield approximately 5000distinct and well-separated colonies on solid bacteriological mediacontaining L-broth containing ampicillin at 100 μg/ml and agar at 1.5%in a 150 mm petri dish when grown overnight at 37° C. Other knownmethods of obtaining distinct, well-separated colonies can also beemployed.

[0078] Standard colony hybridization procedures can then be used totransfer the colonies to nitrocellulose filters and lyse, denature andbake them. Highly stringent conditions include those that are at leastas stringent as, for example, 1×SSC at 65° C., or 1×SSC and 50%formamide at 42° C. Moderate stringency conditions include those thatare at least as stringent as, for example, 4×SSC at 65° C., or 4×SSC and50% formamide at 42° C. Reduced stringency conditions can include, forexample, those that are at least as stringent as 4×SSC at 50° C., or6×SSC and 50% formamide at 40° C.

[0079] The filter is then preferably incubated at 65° C. for 1 hour withgentle agitation in 6×SSC (20× stock is 175.3 g NaCl/liter, 88.2 g Nacitrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mmfilter). The probe can then added to the hybridization mix at aconcentration greater than or equal to 1×10⁶ dpm/mL. The filter is thenpreferably incubated at 65° C. with gentle agitation overnight. Thefilter is then preferably washed in 500 mL of 2×SSC/0.5% SDS at roomtemperature without agitation, preferably followed by 500 mL of2×SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. Athird wash with 0.1×SSC/0.5% SDS at 65° C. for 30 minutes to 1 hour isoptional. The filter is then preferably dried and subjected toatitoradiography for sufficient time to visualize the positives on theX-ray film. Other known hybridization methods can also be employed. Thepositive colonies are then picked, grown in culture, and plasmid DNAisolated using standard procedures. The clones can then be verified byrestriction analysis, hybridization analysis, or DNA sequencing.

[0080] Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences may be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but also onfactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (T_(m)) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the T_(m) of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

[0081] High stringency conditions can employ hybridization at either (1)1×SSC (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedsalmon sperm DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2mg/ml denatured salmon sperm DNA at 42° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedsalmon sperm DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl(pH 7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured salmon spermDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured salmon sperm DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured salmon sperm DNA at42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na₂EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA-DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5-10° C. below that of the calculated T_(m) of the hybrid, where T_(m)in ° C.=(2× the number of A and T bases)+(4× the number of G and Cbases). For hybrids believed to be about 18 to about 49 base pairs inlength, the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

[0082] Moderate stringency conditions can employ hybridization at either(1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/mldenatured salmon sperm DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS,0.1-2 mg/ml denatured salmon sperm DNA at 42° C., (3) 1% bovine serumalbumen (fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 MNaHPO₄=134 g Na₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2mg/ml denatured salmon sperm DNA at 65° C., (4) 50% formamide, 5×SSC,0.02 M Tris-HCl (pH 7.6), 1× Denhardt's solution (100×=10 g Ficoll 400,10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), waterto 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured salmonsperm DNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100μg/ml denatured salmon sperm DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured salmon sperm DNA at42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. Theabove conditions are intended to be used for DNA-DNA hybrids of 50 basepairs or longer. Where the hybrid is believed to be less than 18 basepairs in length, the hybridization and wash temperatures should be 5-10°C. below that of the calculated T_(m) of the hybrid, where T_(m) in °C.=(2× the number of A and T bases)+(4× the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

[0083] Low stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedsalmon sperm DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2mg/ml denatured salmon sperm DNA at 40° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedsalmon sperm DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl(pH 7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured salmon spermDNA at 40° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured salmon sperm DNA at 50° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured salmon sperm DNA at40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C.,or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na₂EDTA, 40 mMNaHPO₄ (pH 7.2), 5% SDS. The above conditions are intended to be usedfor DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-110° C. below that of the calculated T_(m)of the hybrid, where T_(m) in ° C.=(2× the number of A and T bases)+(4×the number of G and C bases). For hybrids believed to be about 18 toabout 49 base pairs in length, the T_(m) in ° C=(81.5°C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na⁺), and “L” is the length of thehybrid in base pairs.

[0084] The invention also encompasses the use of wild type or mutantversions of Gab2 as inserts for vectors. Such vectors can be used toproduce Gab2 proteins in large quantities. They can also be used for thedelivery of nucleic acids to a cell, e.g., a host cell. Such a vectormay also bring about the replication and/or expression of thetransferred nucleic acid pieces and can be used to produce Gab2 proteinin large quantities. Examples of vectors include nucleic acid moleculesderived, e.g., from a plasmid, bacteriophage, or a mammalian, plant orinsect virus, or non-viral vectors such as ligand-nucleic acidconjugates, liposomes, or lipid-nucleic acid complexes. It may bedesirable that the transferred nucleic molecule is operatively linked toan expression control sequence to form an expression vector capable ofexpressing the transferred nucleic acid. Such transfer of nucleic acidsis generally called “transformation,” and refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion. For example, direct uptake, transduction orf-mating are included. The exogenous polynucleotide may be maintained asa non-integrated vector, for example, a plasmid, or alternatively, maybe integrated into the host genome. “Operably linked” refers to asituation wherein the components described are in a relationshippermitting them to function in their intended manner, e.g., a controlsequence “operably linked” to a coding sequence is ligated in such amanner that expression of the coding sequence is achieved underconditions compatible with the control sequence. A “coding sequence” isa polynucleotide sequence which is transcribed into mRNA and translatedinto a polypeptide when placed under the control of (e.g., operablylinked to) appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. Suchboundaries can be naturally-occurring, or can be introduced into oradded the polynucleotide sequence by methods known in the art. A codingsequence can include, but is not limited to, mRNA, cDNA, and recombinantpolynucleotide sequences.

[0085] The vector into which the cloned polynucleotide is cloned may bechosen because it functions in a prokaryotic, or alternatively, it ischosen because it functions in a eukaryotic organism. Two examples ofvectors which allow for both the cloning of a polynucleotide encodingthe Gab2 protein, and the expression of this protein from thepolynucleotides, are the pET22b and pET28(a) vectors (Novagen, Madison,Wis., USA) and a modified pPICZαA vector (InVitrogen, San Diego, Calif.,USA), which allow expression of the protein in bacteria and yeast,respectively. See for example, WO 99/29878, the entire teachings whichare hereby incorporated by reference.

[0086] Once a polynucleotide has been cloned into a suitable vector, itcan be transformed, or transfected, into an appropriate host cell. By“host cell” is meant a cell which has been or can be used as therecipient of transferred nucleic acid by means of a vector. Host cellscan be prokaryotic or eukaryotic, mammalian, plant, or insect, and canexist as single cells, or as a collection, e.g., as a culture, or in atissue culture, or in a tissue or an organism. Host cells can also bederived from normal or diseased tissue from a multicellular organism,e.g., a mammal. “Host cell”, as used herein, is intended to include notonly the original cell which was transformed with a nucleic acid, butalso descendants of such a cell, which still, comprise, or contain, thenucleic acid.

[0087] In one embodiment, the isolated polynucleotide encoding the Gab2protein additionally comprises a polynucleotide linker encoding apeptide. Such linkers are known to those of skill in the art and, forexample the linker can comprise at least one additional codon encodingat least one additional amino acid. Typically the linker comprises oneto about twenty or thirty amino acids. The polynucleotide linker istranslated, as is the polynucleotide encoding the Gab2 protein,resulting in the expression of a GAb2 protein with at least oneadditional amino acid residue at the amino or carboxyl terminus of theprotein. Importantly, the additional amino acid, or amino acids, do notcompromise the activity of the Gab2 protein.

[0088] After inserting the selected polynucleotide into the vector, thevector is transformed into an appropriate prokaryotic strain and thestrain is cultured (e.g., maintained) under suitable culture conditionsfor the production of the biologically active GAb2 protein, therebyproducing a biologically active Gab2 protein, or mutant, derivative,fragment or fusion protein thereof. In one embodiment, the inventioncomprises cloning of a polynucleotide encoding a Gab2 protein into thevectors pET22b, pET17b or pET28a, which are then transformed intobacteria. The bacterial host strain then expresses the Gab2 protein.

[0089] In another embodiment of the present invention, the eukaryoticvector comprises a modified yeast vector. One method is to use a pPICzαplasmid wherein the plasmid contains a multiple cloning site. Themultiple cloning site has inserted into it a HA.Tag motif. Additionallythe vector can be modified to add a NdeI site, or other suitablerestriction sites. Such sites are well known to those of skill in theart.

[0090] One method of producing Gab2, for example, is to amplify thepolynucleotide of SEQ ID NO:6, and clone it into an expression vector,transform the vector containing the polynucleotide into a host cellcapable of expressing the polypeptide encoded by the polynucleotide,culturing the transformed host cell under culture conditions suitablefor expressing the protein, and then extracting and purifying theprotein from the culture.

[0091] In another embodiment, the Gab2 protein may also be expressed asa product of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, sheep or pigs, or as a product of a transgenicplant, e.g., combined or linked with starch molecules in maize. Thesemethods can also be used with a subsequence of SEQ ID NO:6 to produceportions of the protein of SEQ ID NO:5.

[0092] Gab2 may also be produced by conventional, known methods ofchemical synthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed Gab2 protein sequence, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with e.g., recombinantly-produced Gab2, may possessbiological properties in common therewith, including biologicalactivity. Thus, the synthetically-constructed Gab2 protein sequence maybe employed as biologically active or immunological substitute for e.g.,recombinantly-produced, purified Gab2 protein in screening oftherapeutic compounds and in immunological processes for the developmentof antibodies.

[0093] Polynucleotides encoding Gab2 can be cloned out of isolated DNAor a cDNA library. Nucleic acids and polypeptides referred to herein as“isolated” are nucleic acids or polypeptides substantially free (i.e.,separated away from) the material of the biological source from whichthey were obtained (e.g., as exists in a mixture of nucleic acids or incells), which may have undergone further processing. “Isolated” nucleicacids or polypeptides include nucleic acids or polypeptides obtained bymethods described herein, similar methods, or other suitable methods,including essentially pure nucleic acids or polypeptides, nucleic acidsor polypeptides produced by chemical synthesis, by combinations ofchemical or biological methods, and recombinantly produced nucleic acidsor polypeptides which are isolated. An isolated polypeptide thereforemeans one which is relatively free of other proteins, carbohydrates,lipids, and other cellular components with which it is normallyassociated. An isolated nucleic acid is not immediately contiguous with(i.e., covalently linked to) both of the nucleic acids with which it isimmediately contiguous in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term, therefore, includes,for example, a nucleic acid which is incorporated into a vector (e.g.,an autonomously replicating virus or plasmid), or a nucleic acid whichexists as a separate molecule independent of other nucleic acids such asa nucleic acid fragment produced by chemical means or restrictionendonuclease treatment.

[0094] An extracellular stimulus is any substance (e.g., a compound suchas a molecule) that associates or interacts with a cell, directly orindirectly, such that the association or interaction results in asignaling cascade within the cell. Such extracellular stimuli includecytokines, growth factors, hormones and antigens.

[0095] In another embodiment, the biological function of Gab2 can becompromised by inhibitors which can specially block the interaction ofGab2 with its associated molecules. Such inhibitors may be useful in thetreatment of numerous disorders, including allergic responses,immunodisorders and cancer. For example, Gab2 is constitutively tyrosinephosphorylated in a variety of cells transformed by BCR-ABL, theoncogene responsible for chronic myelogenous leukemia, as well as by itsrelative, the TEL-ABL fusion protein, suggesting that deregulation of acascade involving SHP-2 and Gab2 may contribute to cell transformation.In addition, over-expression and/or constitutive phosphorylation of Gab2could contribute to other diseases including other neoplastic diseases.

[0096] As used herein, the term “inhibitor of Gab2 interaction/function”refers to an agent (e.g., an oligonucleotide, a molecule, a compound, ora protein) which can inhibit a (i.e., one or more) function of Gab2.Inhibition can be partial or complete. For example, an inhibitor of Gab2function can inhibit Gab2's association with one or more proteins (e.g.,SHP-2, p85, Grb2) to Gab2 and/or inhibit signal transduction mediatedthrough Gab2 (e.g., MAPK activation, c-fos gene transcription).Accordingly, Gab2-mediated processes and cellular responses (e.g.,proliferation, migration, chemotactic responses, secretion ordegranulation (e.g., acute rejection, chronic rejection)) can beinhibited with an inhibitor of Gab2 function. As used herein, “Gab2”refers to naturally occurring Gab2 (including vertebrate Gab2, e.g.,mammalian Gab2, such as human (Homo sapiens) Gab2) and also encompassesnaturally occurring variants, such as allelic variants and splicevariants.

[0097] Preferably, the inhibitor of Gab2 function is a compound whichis, for example, a small organic molecule, natural product, protein(e.g., antibody, cytokine, antigen), peptide or peptidomimetic.Inhibitors of Gab2 function can be identified, for example, by screeninglibraries or collections of molecules, such as, the Chemical Repositoryof the National Cancer Institute, as described herein or using othersuitable methods.

[0098] The term “natural product”, as used herein, refers to a compoundwhich can be found in nature, for example, naturally occurringmetabolites of marine organisms (e.g., tunicates, algae), plants orother organisms and which possess biological activity, e.g., can inhibitGab2 function.

[0099] Natural products can be isolated and identified by suitablemeans. For example, a suitable biological source (e.g., vegetation) canbe homogenized (e.g., by grinding) in a suitable buffer and clarified bycentrifugation, thereby producing an extract. The resulting extract canbe assayed for the capacity to inhibit Gab2 function, for example, bythe assays described herein. Extracts which contain an activity thatinhibit Gab2 function can be further processed to isolate the Gab2inhibitor by suitable methods, such as, fractionation (e.g., columnchromatography (e.g., ion exchange, reverse phase, affinity), phasepartitioning, fractional crystallization) and assaying for biologicalactivity. Once isolated the structure of a natural product can bedetermined (e.g., by nuclear magnetic resonance (NMR)) and those ofskill in the art can devise a synthetic scheme for synthesizing thenatural product. Thus, a natural product can be isolated (e.g.,substantially purified) from nature or can be fully or partiallysynthetic. A natural product can be modified (e.g., derivatized) tooptimize its therapeutic potential. Thus, the term “natural product”, asused herein, includes those compounds which are produced using standardmedicinal chemistry techniques to optimize the therapeutic potential ofa compound which can be isolated from nature.

[0100] A “neoplastic disorder”, or “Cancer” means neoplastic growth,hyperplastic or proliferative growth or a pathological state of abnormalcellular development and includes solid tumors, non-solid tumors, andany abnormal cellular proliferation, such as that seen in leukemia. Asused herein, “cancer” also means Gab2-dependent cancers and tumors,i.e., tumors that require for their growth (expansion in volume and/ormass) an amplification/overexpression of Gab2. “Regression” refers tothe reduction of tumor mass and size as determined using methodswell-known to those of skill in the art.

[0101] The term “neoplastic disease”, as used herein, refers to amalignant pathologies involving a Gab2-mediated injury or othermalignancies involving Gab2, such as, but not limited to, breast cancer,prostate cancer, carcinomas, such as Ductal Carcinoma in situ, andleukemias (acute, chronic myelocytic, chronic lymphocytic and/ormyelodyspastic syndrome); and lymphomas (Hodgkin's and non-Hodgkin'slymphomas, such as malignant lymphomas (Burkitt's lymphoma or Mycosisfungoides)).

[0102] The term “allergic response”, as used herein, refers toinflammatory or allergic diseases and conditions, including, but notlimited to, respiratory allergic diseases such as asthma, allergicrhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis,interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis,or ILD associated with rheumatoid arthritis, systemic lupuserythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren'ssyndrome, polymyositis or dermatomyositis); systemic anaphylaxis orhypersensitivity responses, drug allergies (e.g., to penicillin,cephalosporins), insect sting allergies; inflammatory bowel diseases,such as Crohn's disease and ulcerative colitis; spondyloarthropathies;scleroderma; psoriasis and inflammatory dermatoses such as dermatitis,eczema, atopic dermatitis, allergic contact dermatitis, urticaria;vasculitis (e.g., necrotizing, cutaneous, and hypersensitivityvasculitis).

[0103] The term “immune disorder”, as used herein, refers to any immunedisease, including autoimmune diseases including, but not limited to,arthritis (e.g., rheumatoid arthritis, juvenile rheumatoid arthritis,psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus,myasthenia gravis, juvenile onset diabetes, nephritides such asglomerulonephritis, autoimmune thyroiditis, Behcet's disease, graftrejection (e.g., in transplantation); or to other diseases or conditions(including Gab2-mediated diseases or conditions), in which undesirableinflammatory responses are to be inhibited can be treated, including,but not limited to, reperfusion injury, atherosclerosis, certainhematologic malignancies, cytokine-induced toxicity (e.g., septic shock,endotoxic shock), polymyositis, dermatomyositis.

[0104] In another embodiment, the nucleic acid sequence of Gab2 of theinvention or its homologs, fragments or complementary sequences can beused to design antisense oligonucleotides. Such agents can beadministered via a variety of routes, including nasal, systemic ortopical. In one embodiment, they can be used in anti-allergy therapy.

[0105] The agent (e.g., Gab2 inhibitor, or additional therapeutic agent)can be administered by any suitable parenteral or nonparenteral route,including, for example, topically (e.g., cream, ointment), or nasally(e.g., solution, suspension). Parenteral administration can include, forexample, intramuscular, intravenous, intraarticular, intraarterial,intrathecal, subcutaneous, or intraperitoneal administration. The agent(e.g., Gab2 inhibitor, additional therapeutic agent) can also beadministered orally (e.g., in capsules, suspensions, tablets ordietary), transdermally, topically, by inhalation (e.g., intrabronchial,intranasal, oral inhalation or intranasal drops) or rectally.Administration can be local or systemic as indicated. The preferred modeof administration can vary depending upon the particular agent (e.g.,Gab2 inhibitor, additional therapeutic agent) chosen, however, oral,systemic or parenteral administration is generally preferred.

[0106] Delivery can be in vitro, in vivo, or ex vivo. Delivery can bevia a variety of means, including transfection, transformation andelectroporation. The cell can be present in a biological sample obtainedfrom the patient (e.g., blood, bone marrow) and used in the treatment ofdisease such as cancer, immunosuppression/immunostimulation,neurodegeneration or cardiac hypertrophy, or can be obtained from cellculture and used to dissect cell proliferation, cell death or proteindegradation cascades in in vivo and in vitro systems. After contact withthe viral vector comprising Gab2 or a Gab2 mutant, the sample can bereturned or readministered to a cell or patient according to methodsknown to those practiced in the art. In the case of delivery to apatient or experimental animal model (e.g., rat, mouse, monkey,chimpanzee), such a treatment procedure is sometimes referred to as exvivo treatment or therapy. Frequently the cell is targeted from thepatient or animal and returned to the patient or animal once contactedwith the viral vector comprising the activated mutant of the presentinvention. Ex vivo gene therapy has been described, for example, inKasid, et al., Proc. Natl. Acad. Sci. USA 87:473 (1990); Rosenberg, etal., New Engl. J. Med. 323:570 (1990); Williams, et al., Nature 310476(1984); Dick, et al., Cell 42.71 (1985); Keller, et al., Nature 318:149(1985) and Anderson, et al., U.S. Pat. No. 5,399,346 (1994).

[0107] Use of timed release or sustained release delivery systems arealso included in the invention. Such systems are highly desirable insituations where surgery is difficult or impossible, e.g., patientsdebilitated by age or the disease course itself, or where therisk-benefit analysis dictates control over cure.

[0108] A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polyproteins,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

[0109] The agent (e.g., Gab2 inhibitor, additional therapeutic agent)can be administered as a neutral compound or as a salt or esther. Saltsof compounds containing an amine or other basic group can be obtained,for example, by reacting with a suitable organic or inorganic acid, suchas hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid andthe like. Compounds with a quaternary ammonium group also contain acounteranion such as chloride, bromide, iodide, acetate, perchlorate andthe like. Salts of compounds containing a carboxylic acid or otheracidic functional group can be prepared by reacting with a suitablebase, for example, a hydroxide base. Salts of acidic functional groupscontain a countercation such as sodium, potassium and the like.

[0110] As used herein, the terms “pharmaceutically acceptable,”“physiologically tolerable” and grammatical variations thereof as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal with a minimum of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike. The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art and need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

[0111] The inhibitor of Gab2 function can be administered to theindividual as part of a pharmaceutical composition comprising a Gab2inhibitor and a pharmaceutically or physiologically acceptable carrier.Pharmaceutical compositions for co-therapy can comprise an inhibitor ofGab2 function and one or more additional therapeutic agents. Aninhibitor of Gab2 function and an additional therapeutic agent can becomponents of separate pharmaceutical compositions which can be mixedtogether prior to administration or administered separately. Formulationwill vary according to the route of administration selected (e.g.,solution, emulsion, capsule).

[0112] The active (e.g., therapeutic) ingredient can be mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient and in amounts suitable for use in the therapeuticmethods described herein. Suitable pharmaceutical or physiologicalcarriers can contain inert ingredients which do not interact with theinhibitor of Gab2 function and/or additional therapeutic agent. Standardpharmaceutical formulation techniques can be employed, such as thosedescribed in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa. Suitable carriers, e.g., excipients for parenteraladministration, include, for example, sterile water, dextrose, glycerol,ethanol, physiological saline, bacteriostatic saline (saline containingabout 0.9% benzyl alcohol), phosphate-buffered saline, Hank's solution,Ringer's-lactate and the like and combinations thereof. Methods forencapsulating compositions (such as in a coating of hard gelatin orcyclodextran) are known in the art (Baker, et al., “Controlled Releaseof Biological Active Agents”, John Wiley and Sons, 1986). In addition,if desired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

[0113] As used herein, the term “effective amount” also means the totalamount of each active component of the composition or method that issufficient to show a meaningful patient benefit, i.e., treatment,healing, prevention or amelioration of the relevant medical condition,or an increase in rate of treatment, healing, prevention or ameliorationof such conditions. For example, an “effective amount” of a Gab2inhibitor is an amount sufficient to achieve a desired therapeuticand/or prophylactic effect, such as an amount sufficient to inhibit mastcell degranulation. In addition, an effective amount is an amountsufficient to inhibit a (i.e., one or more) function of Gab2 (e.g., Gab2extracellular signal-induced c-fos activation, and/or Gab2ligand-induced secretion (e.g. degranulation) of antihistamines), andthereby, inhibit a Gab2-mediated injury (e.g., allergic response, immunedisorder, neoplastic disease). An “effective amount” of an additionaltherapeutic agent is an amount sufficient to achieve a desiredtherapeutic and/or prophylactic effect. When applied to a combination,the term refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously.

[0114] The amount of agent (e.g., Gab2 inhibitor, additional therapeuticagent) administered to the individual will depend on the characteristicsof the individual, such as general health, age, sex, body weight andtolerance to drugs as well as the degree, severity and type ofrejection. The skilled artisan will be able to determine appropriatedosages depending on these and other factors.

[0115] The dosage of the agent of Gab2 of the present invention willalso depend on the disease state or condition being treated along withthe above clinical factors and the route of administration of thecompound. For treating humans or animals, about 10 mg/kg of body weightto about 20 mg/kg of body weight of the protein can be administered. Incombination therapies, e.g., the agents of the invention in combinationwith radiotherapy, chemotherapy, or immunotherapy, it may be possible toreduce the dosage, e.g., to about 0.1 mg/kg of body weight to about 0.2mg/kg of body weight. Depending upon the half-life of the agent in theparticular animal or human, the agent can be administered betweenseveral times per day to once a week. It is to be understood that thepresent invention has application for both human and veterinary use. Themethods of the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time. In addition, the agent can be administered in conjunction withother forms of therapy, e.g., chemotherapy, radiotherapy, orimmunotherapy.

[0116] The term “unit dose” when used in reference to a therapeuticcomposition of the present invention refers to physically discrete unitssuitable as unitary dosage for the subject, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent;i.e., carrier or vehicle. Preferred unit dosage formulations are thosecontaining a daily dose or unit, daily sub-dose, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition to the ingredients, particularly mentionedabove, the formulations of the present invention may include otheragents conventional in the art having regard to the type of formulationin question. Optionally, cytotoxic agents may be incorporated orotherwise combined with the agent, or biologically functional proteinfragments thereof, to provide dual therapy to the patient.

[0117] In addition, Gab2 nucleic acid sequences (e.g., cDNA sequence)can be used to identify relevant downstream targets of Gab2, forexample, those required for allergic responses. Moreover, Gab2 nucleicacid sequences (e.g., cDNA sequence) along with critical bindingmolecule(s), can be used to screen for small molecule and/or naturalproduct inhibitors of the interaction(s).

[0118] Conversely, defects in Gab2 expression and/or sequence maycontribute to other diseases, such as immunodeficiency. Thus, the Gab2coding sequence may also be useful for screening in various diseases,and in gene therapy applications.

[0119] In another embodiment, the novel scaffolding molecule Gab2 can beused to identify inhibitors of PH domain/lipids; Gab2/receptor (e.g.,cytokine, growth factor, hormone and antigen receptor); Gab2/SH2 or SH3protein interactions. Thus, inhibitors which can block Gab2 interactionwith other intracellular molecules via PH domain, SH2, and SH3 domaincan be potentially useful for immunosuppression and cancer therapy.Nucleic acid probes for Gab2 can also be used to detect upregulation ordownregulation of Gab2 product in specimens from patients with leukemiasuch as CML, or to generate antibodies to detect changes in Gab2expression or phosphorylation, for example, in patients with variousneoplastic states.

[0120] RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391, 806-811 (1998)). dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and is a tool for studying gene function.

[0121] RNA interference can be used as a method for knocking down(partially or completely) (out) Gab2. This method of knocking out Gab2gene expression can be used therapeutically or for research (e.g., togenerate models of disease states, to examine the function of a Gab2, toassess whether an agent acts on a Gab2, to validate targets for drugdiscovery). In those instances in which Gab2 function is eliminated, theresulting cell or organism can also be referred to as a knockout. Oneembodiment of the method of producing knockdown cells and organismscomprises introducing into a cell or organism in which Gab2 is to beknocked down, RNA of sufficient length that targets Gab2 and maintainingthe resulting cell or organism under conditions under which RNAi occurs,resulting in degradation of the mRNA of Gab2, thereby producingknockdown cells or organisms. Gab2 knockdown cells and organismsproduced by the present method are also the subject of this invention.

[0122] Furthermore, RNAi can be used as a method of examining orassessing the function of Gab2 in a cell or organism. In one embodiment,RNA of sufficient length which targets mRNA of Gab2 is introduced into acell or organism in which RNAi occurs. The cell or organism is referredto as a test cell or organism. The test cell or organism is maintainedunder conditions under which degradation of Gab2 mRNA occurs. Thephenotype of the test cell or organism is then observed and compared tothat of an appropriate control cell or organism, such as a correspondingcell or organism that is treated in the same manner except that Gab2 isnot targeted. A difference between the phenotypes of the test andcontrol cells or organisms provides information about the function ofthe degraded Gab2 mRNA. The information provided may be sufficient toidentify (define) the function of Gab2 or may be used in conjunctionwith information obtained from other assays or analyses to do so.

[0123] Moreover, RNAi can be used as a method of validating whether anagent acts on Gab2. In this method, RNA of sufficient length thattargets the Gab2 mRNA is introduced into a cell or organism in whichRNAi occurs. Whether the agent has an effect on the cell or organism isdetermined.

[0124] In addition, RNAi can be used as a method of validating whetherGab2 is a target for drug discovery or development. RNA of sufficientlength that targets Gab2 is introduced into a cell or organism. The cellor organism is maintained under conditions in which degradation of theGab2 mRNA occurs, resulting in decreased expression of Gab2. Whetherdecreased expression of Gab2 has an effect on the cell or organism isdetermined, wherein if decreased expression of Gab2 has an effect, thenthe Gab2 product is a target for drug discovery or development.

[0125] RNAi can also be used as a method of treating a disease orcondition associated with the presence of Gab2 protein in an individualcomprising administering to the individual RNA of sufficient lengthwhich targets the mRNA of Gab2 (the mRNA that encodes the protein) fordegradation. As a result, the protein is not produced or is not producedto the extent it would be in the absence of the treatment. Techniquesfor using such methods are found in PCT Application NumberPCT/US01/10188 (WO 01/75164), the contents of which are incorporated byreference herein in their entirety.

[0126] The invention also encompasses genetically manipulated cell andanimals, including knockout and transgenic mice, such as Gab2−/− mice ormice which overexpress Gab2 such as Whey Acidic Promoter-Gab2 (WAP-Gab2)transgene mice as described herein.

[0127] In another embodiment, vectors described herein can be useful ina gene therapy setting, whereby a polynucleotide encoding the Gab2protein, integrins, integrin subunits, or a mutant, fragment, or fusionprotein thereof, is introduced and regulated in a patient. Variousmethods of transferring or delivering DNA to cells for expression of thegene product protein, otherwise referred to as gene therapy, aredisclosed in Gene Transfer into Mammalian Somatic Cells in vivo, N.Yang, Crit. Rev. Biotechn. 12(4):335-356 (1992), which is herebyincorporated in its entirety by reference. Gene therapy encompassesincorporation of DNA sequences into somatic cells or germ line cells foruse in either ex vivo or in vivo therapy. Gene therapy functions toreplace genes, augment or inhibit normal or abnormal gene function, andto combat infectious diseases and other pathologies.

[0128] Strategies for treating these medical problems with gene therapyinclude therapeutic strategies such as identifying the defective geneand then adding a functional gene to either replace or inhibit thefunction of the defective gene or to augment a slightly functional gene;or prophylactic strategies, such as adding a gene for the productprotein that will treat the condition or that will make the tissue ororgan more susceptible to a treatment regimen.

[0129] Many protocols for transfer of the DNA or regulatory sequences ofthe Gab2 protein are envisioned in this invention. Transfection ofpromoter sequences, other than one normally found specificallyassociated with the Gab2 protein, or other sequences which wouldincrease production of the Gab2 protein are also envisioned as methodsof gene therapy.

[0130] Gene transfer methods for gene therapy fall into three broadcategories: physical (e.g., electroporation, direct gene transfer andparticle bombardment), chemical (e.g., lipid-based carriers, or othernon-viral vectors) and biological (e.g., virus-derived vector andreceptor uptake). For example, non-viral vectors may be used whichinclude liposomes coated with DNA. Such liposome/DNA complexes may bedirectly injected intravenously into the patient. It is believed thatthe liposome/IDNA complexes are concentrated in the liver where theydeliver the DNA to macrophages and Kupffer cells. These cells are longlived and thus provide long term expression of the delivered DNA.Additionally, vectors or the “naked” DNA of the gene may be directlyinjected into the desired organ, tissue or tumor for targeted deliveryof the therapeutic DNA.

[0131] Gene therapy methodologies can also be described by deliverysite. Fundamental ways to deliver genes include ex vivo gene transfer,in vivo gene transfer, and in vitro gene transfer. In ex vivo genetransfer, cells are taken from the patient and grown in cell culture.The DNA is transfected into the cells, the transfected cells areexpanded in number and then reimplanted in the patient. In in vitro genetransfer, the transformed cells are cells growing in culture, such astissue culture cells, and not particular cells from a particularpatient. These “laboratory cells” are transfected, the transfected cellsare selected and expanded for either implantation into a patient or forother uses.

[0132] In vivo gene transfer involves introducing the DNA into the cellsof the patient when the cells are within the patient. Methods includeusing virally mediated gene transfer using a noninfectious virus todeliver the gene in the patient or injecting naked DNA into a site inthe patient and the DNA is taken up by a percentage of cells in whichthe gene product protein is expressed. Additionally, the other methodsdescribed herein, such as use of a “gene gun,” may be used for in vitroinsertion of the DNA or regulatory sequences controlling production ofthe Gab2 protein.

[0133] Chemical methods of gene therapy may involve a lipid basedcompound, not necessarily a liposome, to transfer the DNA across thecell membrane. Lipofectins or cytofectins, lipid-based positive ionsthat bind to negatively charged DNA, make a complex that can cross thecell membrane and provide the DNA into the interior of the cell. Anotherchemical method uses receptor-based endocytosis, which involves bindinga specific ligand to a cell surface receptor and enveloping andtransporting it across the cell membrane. The ligand binds to the DNAand the whole complex is transported into the cell. The ligand genecomplex is injected into the blood stream and then target cells thathave the receptor will specifically bind the ligand and transport theligand-DNA complex into the cell.

[0134] Many gene therapy methodologies employ viral vectors to insertgenes into cells. For example, altered retrovirus vectors have been usedin ex vivo methods to introduce genes into peripheral andtumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes,or other somatic cells. These altered cells are then introduced into thepatient to provide the gene product from the inserted DNA.

[0135] Viral vectors have also been used to insert genes into cellsusing in vivo protocols. To direct the tissue-specific expression offoreign genes, WAP-acting regulatory elements or promoters that areknown to be tissue-specific can be used. Alternatively, this can beachieved using in situ delivery of DNA or viral vectors to specificanatomical sites in vivo. For example, gene transfer to blood vessels invivo was achieved by implanting in vitro transduced endothelial cells inchosen sites on arterial walls. The virus infected surrounding cellswhich also expressed the gene product. A viral vector can be delivereddirectly to the in vivo site, by a catheter for example, thus allowingonly certain areas to be infected by the virus, and providing long-term,site specific gene expression. In vivo gene transfer using retrovirusvectors has also been demonstrated in mammary tissue and hepatic tissueby injection of the altered virus into blood vessels leading to theorgans.

[0136] Viral vectors that have been used for gene therapy protocolsinclude but are not limited to, retroviruses, other RNA viruses such aspoliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpesviruses, SV 40, vaccinia and other DNA viruses. Replication-defectivemurine retroviral vectors are the most widely utilized gene transfervectors. Murine leukemia retroviruses are composed of a single strandRNA complexed with a nuclear core protein and polymerase (pol) enzymes,encased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include the gag, pol, and env genes enclosed at by the 5′and 3′ long terminal repeats (LTR). Retroviral vector systems exploitthe fact that a minimal vector containing the 5′ and 3′ LTRs and thepackaging signal are sufficient to allow vector packaging, infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line. Fundamentaladvantages of retroviral vectors for gene transfer include efficientinfection and gene expression in most cell types, precise single copyvector integration into target cell chromosomal DNA, and ease ofmanipulation of the retroviral genome.

[0137] The adenovirus is composed of linear, double stranded DNAcomplexed with core proteins and surrounded with capsid proteins.Advances in molecular virology have led to the ability to exploit thebiology of these organisms to create vectors capable of transducingnovel genetic sequences into target cells in vivo. Adenoviral-basedvectors will express gene product proteins at high levels. Adenoviralvectors have high efficiencies of infectivity, even with low titers ofvirus. Additionally, the virus is fully infective as a cell free virionso injection of producer cell lines is not necessary. Another potentialadvantage to adenoviral vectors is the ability to achieve long termexpression of heterologous genes in vivo.

[0138] Mechanical methods of DNA delivery include fusogenic lipidvesicles such as liposomes or other vesicles for membrane fusion, lipidparticles of DNA incorporating cationic lipid such as lipofectin,polylysine-mediated transfer of DNA, direct injection of DNA, such asmicroinjection of DNA into germ or somatic cells, pneumaticallydelivered DNA-coated particles, such as the gold particles used in a“gene gun,” and inorganic chemical approaches such as calcium phosphatetransfection. Particle-mediated gene transfer methods were first used intransforming plant tissue. With a particle bombardment device, or “genegun,” a motive force is generated to accelerate DNA-coated high densityparticles (such as gold or tungsten) to a high velocity that allowspenetration of the target organs, tissues or cells. Particle bombardmentcan be used in in vitro systems, or with ex vivo or in vivo techniquesto introduce DNA into cells, tissues or organs. Another method,ligand-mediated gene therapy, involves complexing the DNA with specificligands to form ligand-DNA conjugates, to direct the DNA to a specificcell or tissue.

[0139] It has been found that injecting plasmid DNA into muscle cellsyields high percentage of the cells which are transfected and havesustained expression of marker genes. The DNA of the plasmid may or maynot integrate into the genome of the cells. Non-integration of thetransfected DNA would allow the transfection and expression of geneproduct proteins in terminally differentiated, non-proliferative tissuesfor a prolonged period of time without fear of mutational insertions,deletions, or alterations in the cellular or mitochondrial genome.Long-term, but not necessarily permanent, transfer of therapeutic genesinto specific cells may provide treatments for genetic diseases or forprophylactic use. The DNA could be reinjected periodically to maintainthe gene product level without mutations occurring in the genomes of therecipient cells. Non-integration of exogenous DNAs may allow for thepresence of several different exogenous DNA constructs within one cellwith all of the constructs expressing various gene products.

[0140] Electroporation for gene transfer uses an electrical current tomake cells or tissues susceptible to electroporation-mediated mediatedgene transfer. A brief electric impulse with a given field strength isused to increase the permeability of a membrane in such a way that DNAmolecules can penetrate into the cells. This technique can be used in invitro systems, or with ex vivo or in vivo techniques to introduce DNAinto cells, tissues or organs.

[0141] Carrier mediated gene transfer in vivo can be used to transfectforeign DNA into cells. The carrier-DNA complex can be convenientlyintroduced into body fluids or the bloodstream and thensite-specifically directed to the target organ or tissue in the body.Both liposomes and polycations, such as polylysine, lipofectins orcytofectins, can be used. Liposomes can be developed which are cellspecific or organ specific and thus the foreign DNA carried by theliposome will be taken up by target cells. Injection of immunoliposomesthat are targeted to a specific receptor on certain cells can be used asa convenient method of inserting the DNA into the cells bearing thereceptor. Another carrier system that has been used is theasialoglycoportein/polylysine conjugate system for carrying DNA tohepatocytes for in vivo gene transfer.

[0142] The transfected DNA may also be complexed with other kinds ofcarriers so that the DNA is carried to the recipient cell and thenresides in the cytoplasm or in the nucleoplasm. DNA can be coupled tocarrier nuclear proteins in specifically engineered vesicle complexesand carried directly into the nucleus.

[0143] Gene regulation of the Gab2 may be accomplished by administeringcompounds that bind to the Gab2 gene, or control regions associated withthe Gab2 gene, or its corresponding RNA transcript to modify the rate oftranscription or translation. Additionally, cells transfected with a DNAsequence encoding the Gab2 protein may be administered to a patient toprovide an in vivo source of those proteins. For example, cells may betransfected with a vector containing a nucleic acid sequence encodingthe Gab2 protein. The transfected cells may be cells derived from thepatient's normal tissue, the patient's diseased tissue, or may benon-patient cells.

[0144] For example, tumor cells removed from a patient can betransfected with a vector capable of expressing the agent of the presentinvention, and re-introduced into the patient. The transfected tumorcells produce levels of the agent in the patient that inhibit Gab2function, thereby, inhibit the growth of the tumor. Patients may behuman or non-human animals. Cells may also be transfected by non-vector,or physical or chemical methods known in the art such aselectroporation, ionoporation, or via a “gene gun.” Additionally, theDNA may be directly injected, without the aid of a carrier, into apatient. In particular, the DNA may be injected into skin, muscle orblood.

[0145] The gene therapy protocol for transfecting the Gab2 agent into apatient may either be through integration of the Gab2 DNA into thegenome of the cells, into minichromosomes or as a separate replicatingor non-replicating DNA construct in the cytoplasm or nucleoplasm of thecell. Expression of the GAb2 protein may continue for a long-period oftime or may be reinjected periodically to maintain a desired level ofthe protein(s) in the cell, the tissue or organ or a determined bloodlevel.

[0146] In one embodiment, the Gab2 peptides of the present invention canbe used to raise antibodies against, e.g., specific for, Gab2. Suchpeptides can be used to immunize or vaccinate an animal. Thus, theinvention encompasses antibodies and antisera, which can be used fortesting of novel GAb2 proteins, and can also be used in diagnosis,prognosis, prevention or treatment of diseases and conditionscharacterized by, or associated with, Gab2 activity or lack thereof.Such antibodies and antisera can also be used to up-regulate ordown-regulate Gab2 where desired.

[0147] Such antibodies and antisera can be combined withpharmaceutically-acceptable compositions and carriers to formdiagnostic, prognostic or therapeutic compositions. The term “antibody”or “antibody molecule” refers to a population of immunoglobulinmolecules and/or immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antibody combining site orparatope.

[0148] Passive antibody therapy using antibodies that specifically bindthe Gab2 protein can be employed to modulate Gab2-mediated, e.g.,Gab2-dependent, processes such as degranulation, cytokine geneexpression, and MAPK activation. In addition, antisera directed to theFab regions of antibodies of the Gab2 protein can be administered toblock the ability of endogenous antisera to the proteins to bind theproteins.

[0149] The antibodies of the present invention can be either polyclonalantibodies or monoclonal antibodies. These antibodies that specificallybind to the Gab2 proteins or with a protein that associates with Gab2can be used in diagnostic methods and kits that are well known to thoseof ordinary skill in the art to detect or quantify the Gab2 proteins ina body fluid or tissue. Results from these tests can be used to diagnoseor predict the occurrence or recurrence of a cancer and otherGab2-mediated diseases.

[0150] The invention also includes use of the Gab2 protein, antibodiesto this protein, and compositions comprising this protein and/or itsantibodies in diagnosis or prognosis of diseases characterized by Gab2activity. As used herein, the term “prognostic method” means a methodthat enables a prediction regarding the progression of a disease of ahuman or animal, e.g., a mammal, diagnosed with the disease, inparticular, an Gab2-dependent disease. The term “diagnostic method” asused herein means a method that enables a determination of the presenceor type of Gab2-dependent disease in or on a human or animal.

[0151] The Gab2 protein can be used in a diagnostic method and kit todetect and quantify antibodies capable of binding the protein. Thesekits would permit detection of circulating antibodies to the Gab2protein. Patients that have such circulating anti-Gab2 proteinantibodies may be more likely to have Gab2-related disorders, such ascancers, immune disorders or allergic disorders, and may be more likelyto have recurrences of cancer after treatments or periods of remission.

[0152] In one embodiment, the Fab fragments of these anti-Gab2 proteinantibodies may be used as antigens to generate anti-Gab2 proteinFab-fragment antisera which can be used to neutralize anti-Gab2 proteinantibodies to treat certain disorders caused by a decrease in Gab2activity, e.g., that are caused by Gab2 underexpression, for example,immune disorder. Such a method would reduce the removal of circulatingprotein by anti-Gab2 protein antibodies.

[0153] The present invention is further illustrated by the followingexamples, which are not intended to be limiting in any way.

EXAMPLES Example 1 Purification and Sequencing of Gab2

[0154] Cell Culture

[0155] Cell lines were grown in RPMI/10% FCS and the appropriatecytokine/growth factors.

[0156] Northern Blotting

[0157] Blots containing mouse tissue poly(A) RNA (Clontech, Palo Alto,Calif.) or total RNA (10 μg) from murine hematopoietic cells (providedby Dr. D. Zhang, Beth Israel Deaconess Medical Center, Boston, Mass.)were hybridized to radiolabelled cDNA probes, as indicated.

[0158] Purification and Sequencing

[0159] Gab2 was purified from ˜5×10¹⁰ P210BCR-ABL BaF3 cells.Affinity-purified rabbit anti-SHP-2 antibodies (2.6 mg) were crosslinkedonto protein A Sepharose beads (Harlow, E. and Lane, D., Antibodies: ALaboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory) (1988)) using dimethylpimelimidate (Pierce, Rockford, Ill.).P210 BCR-ABL BaF3 cells (4×10) were resuspended in 40 ml hypotonicbuffer (HB) containing protease and phosphatase inhibitors (Timms, J. F.et al., Mol. Cell. Biol. 18, 3838-3850 (1998)) and homogenized. Lysatesclarified at 100,000×g were loaded onto Q-Sepharose and washedsuccessively with HB and 20 mM Tris, pH 7.4/100 mM NaCl. Bound materialwas cluted in 20 mM Tris, pH 7.4/350 mM NaCl, diluted to a solution of20 mM Tris7.4/150 mM NaCl/0.2% NP40, incubated with the anti-SHP-2antibody beads, and washed with five volumes each of 20 mM Tris, pH7.4/500 mM NaCl and 100 mM glycine, pH 6.0. Bound material was eluted in100 mM glycine, pH 2.5, and neutralized with 1M Tris, pH 8.0. Pooledeluates from 10 cycles of this protocol were concentrated (Centricon-30;Amicon, Bedford, Mass.), acetone-precipitated, and resolved by SDS-PAGE.

[0160] A fraction of the final preparation was transferred to a nylonmembrane and stained for total protein (Amersham, Piscataway, N.J.) andwith anti-phosphotyrosine antibodies (anti-pTyr), and the immunoblotswere examined. In addition to SHP-2 and Gab2, co-purifying speciesincluded an 85 kDa band, which was identified as the p85 subunit ofPI-3K, and a band at ˜150 kDa (p 150). The 150 kDa species associatedwith SHP-2 only in some BCR-ABL-transformed hematopoietic cell lines(Gu, H. et al., J. Biol. Chem. 272, 16421-16430 (1997)). Although SHP-2was reported to associate with P210BCR-ABL (Tauchi, T. et al., J. Biol.Chem. 269, 15381-7 (1994)), a ˜210 kDa species was barely detectable inthe final preparation, indicating that it is an association of lowstoichiometry.

[0161] The 97 kDa band (which represented Gab2) was digested withendoprotease Lys-C. The resultant peptides were resolved byreverse-phase HPLC. The HPLC-resolved Lys-C peptides (nine peaks) weresequenced by Edman degradation (FIG. 1), with additional support fromMS/MS sequencing, by the Harvard Microchemistry Facility. These peptides(Table 1) did not match any protein in the database, indicating that the97 kDa band was novel. TABLE 1 List of peptides obtained by Edmansequencing Peptide No. Sequence Location in p97 GK102 (S/G/A) [G][G]*2-4 GK124 [Q]LEED[Y][Y][G][L][S](K)(G) not present PK85 TQALQN[T]-(Q)*641-649 PK91 [D][S]TYDLPR[S]LA* 260-270 GK41(Q/E/P)(I/S)(L/R)(D/H)(N/K)TEFK 251-259 GK49 VD = VQVDK 631-638 GT131ELQDSFVFDIK 81-91 GT142 AKPTPLDLRNNTVIDEL* 512-529 GK95SSLTGSETDNEDVYTFK 277-293

Example 2 Cloning of Gab2

[0162] Reverse transcription-polymerase chain reaction (RT-PCR) was usedto obtain a cDNA fragment corresponding to peptide GT142, and thisfragment was used to clone full length Gab2 cDNAs. Degenerate primerscorresponding to all possible codons for the sequences KAKPTP (SEQ IDNO: 1) and TVIDEL (SEQ ID NO: 2) in peptide GT-142 (Table 1) weresynthesized and used in a RT-PCR reaction with total RNA fromP210BCR-ABL BaF3 cells. The expected 68 bp PCR product was subclonedinto pUC19. Three inserts were sequenced and found to encode GT-142. Aunique sequence (5′CCTTGACCTGAGAAACAACAC3′) (SEQ ID NO: 3) encoding themiddle of GT-142 (LDLRNN)(SEQ ID NO: 4) served as the 5′ primer in a3′RACE reaction (GIBCO-BRL, Rockville, Md.), yielding a single 800 bpproduct. Its sequence revealed a single open reading frame containingpeptides GT142, GK49 and PK85 (FIG. 2; Table 1). The 800 bp product wasused as to probe a BaF3 cell cDNA library in λUniZap (provided by Dr.Alan D'Andrea, Dana Farber Cancer Institute, Boston, Mass.). Twentypositive clones were obtained, the two largest containing ˜5 kb inserts.Plasmids containing cDNAs were recovered by superinfection, according tothe manufacturer's instructions (Stratagene, La Jolla, Calif.).

[0163] The two largest clones contained the consensus Kozak sequencespecifying transnational initiation (GAC ATG AGC), an in-frame upstreamstop codon, and a single long (1998 bp) open reading frame. The sequencepredicted a 666 amino acid protein with a calculated molecular weight of73 kDa (FIG. 2). The nucleotide sequence is deposited as GenBankAccession Number AF104244 (FIG. 6). Eight of nine Lys-C peptidesobtained by microsequencing were found within the predicted sequence(FIG. 2 and Table 1), strongly suggesting that this cDNA encodes Gab2.Presumably, the one missing peptide derived from a trace contaminant.

[0164] This cDNA must encode bona fide Gab2 from BaF3 cells, becausepeptide GK102, comprising the p97 N-terminus, is predicted by this cDNAsequence, although it is absent in the sequence of unknown gene KIAA0571(FIG. 3 and Table 1). KIAA0571 was deposited in GenBank (AccessionNumber AB011143) as part of a collection of human brain cDNAs (Nagase etal., DNA Res. 5, 31-39 (1998)). The KIAA0571 cDNA encoded a protein with88% amino acid identity to Gab2, suggesting that KIAA0571 may be thehuman homolog of Gab2. The nucleotide sequences of the 5′ ends of Gab2and KIAA0571 are highly divergent, such that the first 39 amino acids ofGab2, which comprises part of the PH domain, were absent in KIAA0571. Inthe absence of this sequence information, the likely function(s) of Gab2cannot be deduced. In addition, the function of KIAA0571 open readingframe, were it to be expressed in vivo, is almost certain to becompromised, since PH domains bind to phospholipids and promotetargeting to cellular membranes. KIAA0571 may represent an alternativelyspliced form of Gab2, although cloning artifacts in KIAA0571 cannot beexcluded.

[0165] The Gab2 protein contained an N-terminal PH domain that has apotential Grb2 SH2 domain binding site, followed by a long region withmultiple potential tyrosyl phosphorylation sites, two serine-richstretches (a.a. 126-188 and 532-600, respectively), and 2 PXXP sites,potentially capable of binding SH3 or WW domains (FIGS. 2, 3 and Table2). Mammalian Gab1 and Drosophila Dos have similar topography and somesequence similarity to Gab2 (FIGS. 3, 4). The greatest similarityresides in their PH domains, with 73% identity between Gab2 and Gab 1,and 45% identity between Gab2 and Dos (FIG. 4; top panel). Most of thepotential phosphorylation sites and their relative positions within eachprotein are conserved, as is one of the PXXP motifs (FIG. 3 and Table2). TABLE 2 List of potential SH2 domain binding motifs in Gab2Potential Tyrosyl SH2 Domain Phosphorylation Sites Containing proteinsYKNE Grb2 YSLP PLCγ YDLP Crk or Nck YQIP Crk or PLCγ YEYP Crk YVPM p85(PI3-K) YIPM p85 (PI3-K) YVPM p85 (pI3-K) YLAL SHP-2 YVQV SHP-2

[0166] There are also potentially significant sequence differencesbetween these proteins. The Met binding domain (MBD) of Gab1 (a.a.450-532) mediates association with c-Met/HGFR (Weidner et al., Nature384, 173-176 (1996)). The cognate region in Gab2 (a.a. 443-514) exhibitsonly 36% amino acid identity (FIG. 4, bottom panel). The MBD, of Gab1,but not Gab2, contains two proline-rich stretches that comprisepotential Grb2 SH3 domain-binding sites (Yu, H. et al., Cell 76, 933-945(1994)). Conversely, two potential 14-3-3 binding sites (Yaffe, M. B. etal., Cell 91, 961-971 (1997)) are present only in Gab2 (RKS₁₆₀SAP, andRQS₆₅₈SEP). It remains to be determined whether Gab2 binds 14-3-3proteins in vivo.

Example 3 Confirmation of Gab2 cDNA

[0167] To confirm that the cDNA encoded Gab2, a vector directingexpression of HA-tagged GAb2 construct (Gab2HA) was transientlytransfected into BaF3 cells. BaF3 cells were washed in serum-free RPMI,resuspended at 10⁷ cells/0.5 ml in RPMI/10% FCS, and incubated (10minutes) with the indicated amounts of Gab2 expression vector and/or 20μg of the SHP2 expression vector, the indicated amount of promoterluciferase reporter, and 20 ng of Renilla luciferase-TK reporter(Promega, Madison, Wis.).

[0168] Constructs encoding Gab2 (Gab2HA), Gab2 lacking amino acids604-662 (Gab2ΔY2HA), and Gab2 with tyrosines 604/633 mutated tophenylalanine (Gab2DM), all with C-terminal HA tags, were constructed byPCR, using Gab2 cDNA as the template. PCR products were cloned into pEBB(from Dr. B. Mayer, Children's Hospital, Boston, Mass.), which directedexpression under the control of the elongation factor 1-α promoter. Thetransfected Gab2HA fusion protein, as well as endogenous Gab2, migratedat an apparent mobility of about 97 kDa. Upon IL-3 stimulation, Gab2HAbecame tyrosyl phosphorylated and co-immunoprecipitated with SHP-2,consistent with its expected properties (Gu, H. et al., J. Biol. Chem.272, 16421-16430 (1997)).

[0169] Further evidence was provided by studies with specificantibodies. A fragment encoding Gab2 a.a. 523-666 was subcloned in frameinto pGEX 4T-1 (Pharmacia, Piscataway, N.J.). GST fusion protein wasproduced as described (Lechleider et al., J. Biol. Chem. 268,13434-13438 (1993)). Affinity purified antibodies were prepared bypassing antisera sequentially over GST and GST-Gab2 bound to Affi-Gel 15(Bio-Rad, Hercules, Calif.), prepared as described (Frangioni et al.,Cell 68, 545-560 (1992)). 4 μg of affinity purified antibodiesquantitatively deplete Gab2 from 10⁷ BaF3 cells. Rabbit antibodiesagainst peptide GT142 coupled to KLH were generated by BABCO (Berkeley,Calif.).

[0170] Immunoprecipitations and Immunoblotting.

[0171] Cell lysis, immunoprecipitation, immunoblotting, and detection byenhanced chemi-luminescence (Amersham, Piscataway, N.J.) were performed(Timms et al., Mol. Cell. Biol. 18, 3838-3850 (1998)). Monoclonalantibody 9E10 (against the myc-epitope) was obtained from BABCO(Berkeley, Calif.). Monoclonal anti-phosphotyrosine antibody 4G10 wasobtained from UBI (Santa Cruz). Anti-SHP2 immunoprecipitations utilized1 μg antibody/10⁷ BaF3 cell equivalents. Anti-Gab2 immunoprecipitationsutilized 4 μg antibody/10⁷ BaF3 cell equivalents. Dilutions forimmunoblotting were: anti-SHP-2 (1:2500); anti-Grb-2 (1:1000, SantaCruz), anti-Shc (1:1,000, Transduction Laboratories, Lexington, Ky.);anti-p85 (1:3500, from Dr. C. Carpenter, Beth Israel Deaconess MedicalCenter); anti-MAPK (1:5,000, from Dr. J. Blenis, Harvard Medical School,Boston, Mass.); anti-Gab2 peptide antibodies (1:500); anti-GST-Gab2antibodies (1:2500) and anti-pTyr antibodies (0.5 μg/ml).

[0172] BaF3 cells, starved in RPMI/0.8% BSA for 6 hours, were stimulatedwith recombinant IL-3 (10 ng/ml). Polyclonal antibodies against peptideGT-142 (Table 1) detected a 97 kDa protein in SHP-2 immunoprecipitatesfrom IL-3-stimulated BaF3 cells; this protein co-migrated with the 97kDa phosphotyrosyl protein associated with SHP-2. Antibodies againstGST-Gab2 specifically immunoprecipitated 97 kDa and 70 kDa tyrosylphosphorylated proteins from IL-3-stimulated BaF3 cells; these proteinsco-migrated with the tyrosyl phosphoproteins present in anti-SHP-2immunoprecipitates. Probing these immunoblots with anti-GST-p97antibodies confirmed that the 97 kDa protein immunoprecipitated withanti-Gab2 and anti-SHP-2 antibodies is Gab2.

[0173] These data established that Gab2 is a 97 kDa SHP-2 bindingprotein. To determine whether Gab2 is the only component of the 97 kDatyrosyl phosphorylated band associated with SHP-2 in IL-3-stimulatedBaF3 cells, immunodepletion studies were performed. Quantitativedepletion of Gab2 left no remaining 97 kDa phosphotyrosyl protein(s) inSHP-2 immunoprecipitates. Likewise, nearly all of the Gab2 in BaF3 cellsassociated with SHP-2 upon IL-3 stimulation (as indicated by acomparison of intensities of 97 kDa band in anti-Gab2 and anti-SHP-2lanes), suggesting that SHP-2 probably is critical for signals emanatingfrom Gab2. In contrast, only ˜10% of SHP-2 was found in a complex withGab2 upon IL-3 stimulation. This excess of SHP-2 is consistent with thepossibility that it interacts with additional targets in BaF3 cells.

Example 4 Gab2 Expression and Response to Diverse Hematopoietic Stimuli

[0174] Since a 97 kDa protein was only observed associated with SHP-2 inhematopoietic cells, Gab2 was expected to be hematopoieticcell-specific. Indeed, Gab2 was expressed in many (but not all)hematopoietic cell lines, representing multiple lineages, and was notexpressed in fibroblasts. Surprisingly, however, a 6 kb Gab2 transcriptwas observed in most tissues, with two additional smaller transcriptsfound in testis. Expression of Gab2 was highest in heart, testis, andlung, with lower levels in brain and liver. Although Gab2 participatesin lymphocyte signaling, its expression was relatively low in spleen andthymus. Gab1 expression in the same tissues was largely overlapping,suggesting that Gab1 and Gab2 are co-expressed in at least some celltypes. The relative levels of Gab2 and Gab1 expression are not the samein all tissues (as indicated by a comparison expression in brain, liver,and testis).

[0175] SHP-2 associated with proteins of similar size to Gab2 (95-110kDa) in many signaling cascades, so Gab2 tyrosyl phosphorylation inresponse to a range of stimuli was examined. Kit225 cells (from Dr. P.Brennan, ICRF, London, U.K.), starved in RPMI/10% FCS for 48 hours, werestimulated with IL-2 (25 units/ml). BAC1.2F5 cells were stimulated asdescribed (Timms et al., Mol. Cell. Biol. 18, 3838-3850 (1998)). WEHI231 cells were stimulated with 15 μg/ml goat F(ab)′₂ anti-mouse IgG(Jackson ImmunoResearch Laboratories, West Grove, Pa.). Jurkat cellswere stimulated with 1 μg/ml anti-CD3 antibody, OKT3 (ATCC), andcrosslinked with 10 μg/ml rabbit anti-mouse IgG.

[0176] SHP-2 associated with a 100 kDa p-Tyr protein upon CSF-1stimulation of myeloid progenitor cell lines (Carlberg andRohrschneider, J. Biol. Chem. 272, 15943-15940 (1997)) or macrophages(Timms et al., Mol. Cell. Biol. 18, 3838-3850 (1998)). CSF-1 stimulationof BAC1.2F5 cells resulted in the rapid tyrosyl phosphorylation of Gab2and its association with SHP-2. Similar results were obtained followingIL-2 stimulation of Kit 225 cells or erythropoietin stimulation oferythropoietin receptor-expressing BaF3 cells. In Jurkat cells, TCRstimulation results in association of SHP-2 with a “110 kDa” p-Tyrprotein (Frearson et al., Eur. J. Immunol. 26, 1539-1543 (1996);Frearson and Alexander, J. Exp. Med. 187, 1417-1426 (1998)); in thesecells, Gab2 was rapidly tyrosyl phosphorylated and associated withSHP-2. B cell receptor (BCR) stimulation of WEHI-231 cells also led toGab2 phosphorylation.

Example 5 Association of Gab2 with Other Signaling Molecules

[0177] The ability of Gab2 to also associate with other signalingmolecules was examined. Tyrosyl phosphorylation of Gab2HA occurredwithin 1 minute of IL-3 stimulation of BaF3 cells, and then increasedfurther, peaking by 10 minutes and accompanied by a dramatic decrease inGab2 electrophoretic mobility. Besides SHP-2, tyrosyl phosphorylatedGab2 associated with Shc and the p85 subunit of PI-3K. In contrast,consistent with its PXXP motifs, Gab2 associated constitutively withGrb2. Shc, p85 and Grb2 also co-immunoprecipitated with endogenous Gab2from IL-3-stimulated BaF3 cells. Sites within Gab2 conform to theconsensus for the SH2 domains of Crk (and Crk-II and Crk-L) and PLCy(FIG. 3 and Table 2), but using available immunoreagents, endogenousGab2 or Gab2HA association with these proteins was not detected.Although Grb2 binds Gab2, Sos was not detected in Gab2immunoprecipitates, perhaps because Grb2 binds primarily via its SH3domain to Gab2.

Example 6 Role of Gab2 and the Gab2/SHP-2 interaction in IL-3 Signaling

[0178] The effects of expressing wild type Gab2 and Gab2 mutants unableto bind SHP-2 on IL-3 signaling were examined. Two tyrosines within Gab2(Y₆₀₄LAL/Y₆₃₃VQV) conform to the consensus for binding the SH2 domainsof SHP-2 (Songyang et al., Cell 72, 767-778 (1993)); both sites arehighly phosphorylated. A deletion mutant lacking these residues (a. a.604-666) was generated, and a C-terminal HA tag was appended, asdescribed in Example 5. This mutant (Gab2ΔY2HA) or wild type Gab2HA wastransiently transfected into BaF3 cells.

[0179] Although Gab2ΔY2HA was expressed, it did not bind SHP-2 upon IL-3stimulation. IL-3-induced tyrosyl phosphorylation of Gab2ΔY2HA wassimilar, or even slightly less than that of wild type Gab2HA. Theminimal effect on Gab2 tyrosyl phosphorylation observed upon mutatingits SHP-2 binding sites contrasts with its increased tyrosylphosphorylation upon over-expression of catalytically inactive mutantsof SHP-2. Although C-terminal deletion eliminated SHP-2 binding, Gab2association with p85 and Shc was not decreased, suggesting that Gab2structure was not grossly perturbed in Gab2ΔY2HA. Grb2 binding toGab2ΔY2HA also decreased slightly, particularly upon IL-3 stimulation,most likely because some Grb2 associates indirectly with Gab2 viabinding to tyrosyl phosphorylated SHP-2 (Welham et al., J. Biol. Chem.269, 23764-23768 (1994)).

[0180] SHP-2 is required for induction of c-fos. Therefore, the role ofGab2 in IL-3-induced c-fos promoter activity was assessed. For c-fosreporter assays, 1.5 μg of c-fos promoter (nt −710 to +42)-luciferasereporter were used (Hu et al., Science 268, 100-102 (1995)). STAT-driventransctivation was measured using 1.5 μg of a GAS-luciferase reporter(Jaster et al., Mol. Cell. Biol. 3364-3372 (1997)). For Elk assays,Gal4-Elk-1 (2 μg) and Gal4-luciferase (1 μg) were used (Bennett et al.,Mol. Cell. Biol. 16, 1189-1202 (1996)). Cells were electroporated at300V/800 μF, transferred to fresh RPMI/10% WEHI supernatant/10% FCS and,after three hours, were starved in RPMI/10% FCS. Twelve hourspost-transfection, transfected cells (approximately 10⁶/condition) wereincubated in RPMI/10% FCS alone or with murine IL-3 (1 ng/ml) for twohours. Luciferase assays were performed with a kit (Promega, Madison,Wis.). Promoter activities were normalized to Renilla luciferase levels.

[0181] Over-expression of wild type Gab2HA had little effect onIL-3-stimulated c-fos luciferase activity, although it did evoke a small(˜2-fold), but reproducible, increase in basal activity (FIG. 5A).However, expression of a comparable level of Gab2ΔY2HA decreasedIL-3-evoked c-fos reporter activity (FIG. 5A) in a dose-dependent manner(FIG. 5B). Presumably, Gab2ΔY2HA displaced endogenous Gab2 from itsproper location in vivo but, unable to bind SHP-2, Gab2ΔY2HA cannottransmit a signal necessary for full activation of c-fos. Dominantnegative SHP-2 (SHP2AP) inhibited IL-3 induced c-fos activation (FIG.5A), and the increased basal c-fos luciferase activity evoked by Gab2HAwas blocked by dominant negative SHP-2. A Gab2 mutant in which Y604 andY633 were converted to phenylalanine (Gab2DM) also lacked SHP-2 bindingand inhibited IL-3-evoked c-fos luciferase activity (FIG. 5C). Thesefindings suggest that Gab2 function, and, in particular, Gab2 binding toSHP-2, are required for full cytokine-induced c-fos activation.

[0182] The SRE (serum response element), which binds the SRF/TCF (serumresponse factor/TCF) complex is required for c-fos activation. MAPKphosphorylates Ets family transcription factors that comprise TCF,(e.g., Elk-1), increasing their transactivation potential. The abilityof a Gal4-Elk-1 fusion to drive GAL4-luciferase activity in response toIL-3 was inhibited by Gab2ΔY2HA, indicating Gab2/SHP-2 was required forElk-driven transactivation. STAT 5 also contributed to activation of thec-fos promoter in response to IL-3/GM-CSF (Rajotte et al., Blood 88,2906-2916 (1996)). The Gab2/SHP-2 complex was required for fullactivation of a STAT-responsive element as well. Thus, Gab2/SHP-2association was required for full cytokine-induced activation of the twomajor elements in the c-fos promoter.

[0183] The MEK inhibitor PD98059 ablated IL-3-induced c-fos luciferaseactivity, indicating that MEK/MAPK activation is essential forIL-3-induced c-fos promoter activation in BaF3 cells. Gab2ΔY2HA wasexpected to also inhibit IL-3-induced MAPK activity, particularly sinceSHP-2 is required for cytokine-induced MAPK activation. Transientover-expression of wild type Gab2HA potentiated MAPK activation inresponse to IL-3, suggesting that Gab2 can signal to MAPK. Surprisingly,however, Gab2ΔY2HA or Gab2DM not only failed to inhibit, but potentiatedIL-3-evoked MAPK activation.

[0184] Because of these surprising findings, the role of SHP-2 inIL-3-induced MAPK activation was re-examined. For MAPK assays, BaF3cells (10) were co-transfected with 20 μg Gab2 or SHP2 expressionplasmids and 2 μg of myc-tagged Erk1. Cells (5×10⁶) were stimulated withmurine IL-3 for various times, washed, lysed in NP40 buffer, and Myc-Erkwas immunoprecipitated with 9E10 (1 μl of ascites/sample). Kinase assaysusing myelin basic protein (MBP) were performed as described (Bennett etal., Mol. Cell. Biol. 16, 1189-1202 (1996)).

[0185] As mentioned above, previous work showed that SHP-2 is necessaryfor IL-3-induced MAPK activity. Consistent with these reports, twomutants of SHP-2, SHP2ΔP and SHP2CS inhibited IL-3-induced MAPKactivation. It was concluded that the Gab2/SHP-2 complex was requiredfor full activity of the c-fos promoter, acting on Ets and STAT familytranscription factors via a cascade parallel to MAPK activation, andthat SHP-2 must act at more than one point in cytokine signaling.

Example 7 Characteristics of Gab2−/− BMMCs

[0186] Wild type (WT) and Gab2 knockout (−/−) BMMCs were incubated with2 ug/ml anti-DNP monoclonal IgE (SPE-7) for 1 hour on ice. Cells werewashed and incubated with FITC-anti-IgE rat monoclonal antibody for 30minutes. Cell surface expression of FcεRI were determined by FACS, andfound to be normal in Gab2−/− BMMCs.

[0187] BMMCs were sensitized with anti-DNP IgE, and stimulated with 10ng/ml anti-dinitrophenol (DNP). Cells were lysed, and total cell lysateswere resolved by SDS-PAGE, western blotted with anti-phosphotyrosineantibody (pTyr), and reprobed with anti-Akt antibodies. Total tyrosylphosphorylation was found to be normal in Gab2−/− BMMCs upon FcεRIengagement.

[0188] BMMCs were sensitized with anti-DNP-IgE, stimulated with 10 ng/mlDNP, and lysed. Syk and LAT were immunoprecipitated by anti-Syk and LATantibodies, resolved by SDS-PAGE, and western blotted withanti-phosphotyrosine antibody (pTyr), and reprobed with Syk antibodies.Syk and LAT tyrosyl phosphorylation are normal in Gab2−/− BMMCs uponFcεRI engagement.

[0189] BMMCs were sensitized with DNP mouse IgE (2 ug/ml) for 12 hoursand labeled with ³H-serotonin for 3 hours. Unincorporated label waswashed away and cells were stimulated for 15 minutes with the indicatedconcentrations of DNP. Serotonin released into the media and remainingin the cell pellet was quantified by scintillation counting, and it wasdetermined that FcεRI-mediated degranulation is impaired in Gab2−/−BMMCs.

[0190] BMMCs were sensitized with anti-DNP-IgE, and stimulated with 10ng/ml DNP for 0 and 1 hour. Total RNAs were isolated from BMMCs, andreverse-transcribed into first cDNA by reverse-transcriptase. Therelative level of TNFα and IL-6 cDNAs in each sample was determined byReal time PCR, and Fcε-evoked TNFα and IL-6 gene expression are impairedin Gab2−/− BMMCs.

[0191] BMMCs were sensitized with anti-DNP-IgE, stimulated with 10 ng/mlDNP, and lysed. PLCγl was immunoprecipitated by anti-PLCγl antibodies,resolved by SDS-PAGE, and western blotted with anti-phosphotyrosineantibody (pTyr), and reprobed with anti-PLCγl antibodies. It wasdetermined that tyrosyl phosphorylation of PLCγ is impaired in Gab2−/−BMMCs upon FcεRI crosslinking.

[0192] BMMCs were sensitized with anti-DNP IgE, and stimulated with 10ng/ml DNP. Cells were lysed, and total cell lysates were resolved bySDS-PAGE, western blotted with anti-phospho-Akt antibodies, and reprobedwith anti-Akt antibodies. Akt phosphorylation was found to be impairedin Gab2−/− mast cells upon FcεRI engagement.

[0193] BMMCs were sensitized with anti-DNP IgE, and stimulated with 10ng/ml DNP. Cells were lysed, and total cell lysates were resolved bySDS-PAGE, western blotted with anti-phospho-Akt, phospho-p38, andphospho-MAPK antibodies, and reprobed with anti-Akt and MAPK antibodies.JNK and p38 phosphorylation is defective in Gab2−/−BMMCs, however, MAPKphosphorylation is not affected in Gab2−/−BMMCs.

[0194] Gab2−/−BMMCs were infected with MSCV-IRES-GFP virus expressingwild type Gab2 (WT) or virus alone (Vector). GFP positive BMMCs weresorted out by FACs, expanded, sensitized by IgE, and stimulated with 10ng/ml DNP—HSA. Cells were lysed, and equal amount of total cell lysateswere resolved by SDS-PAGE, transferred, and blotted withanti-phospho-Akt (473), phopho-JNK, phospho-38, and phospho-MAPKrespectively. The same blot was reprobed with anti-Akt and Erk2.Expression of wild type Gab2 rescues signaling defects in Gab2−/−BMMCs.

[0195] 20 ng of anti-DNP-IgE was injected intradermally into one ear ofthe mice. 24 hours later, 100 μg DNP in 200 μl of PBS with 2% Evans Bluewas injected into the tail vein of the mice. 30 minutes later, the micewere sacrificed, and the ears were removed, cut into small pieces. Evansblue dye was extracted from the ears with formamide and 80° C.incubation for 3 hours, and quantified by reading OD at 610 nm. Passivecutaneous anaphylaxis was found to be defective in Gab2−/− mice.

Example 8 Location of Gab2 Gene on Human Chromosome 11q13.3-14.2

[0196] Gab2 was purified and cloned from BCR-ABL transformed cells (Gu,H. et al., Mol Cell. 2, 729-740 (1998)). Unpublished data indicated thatGab2 can be phosphorylated by BCR-ABL in vitro. To further explorepotential Gab2 involvement in human disease, FISH analysis was performedto localize the Gab2 gene on human genome. The Gab2 gene was located onhuman chromosome 11q13.3-14. Importantly, chromosome 11q13 is amplifiedin about 15% of primary breast cancers (Hui, R. et al., Oncogene 15,1617-1623 (1997)). Cyclin D1, also located in the 11q13 amplicon, is oneof the few genes expressing in the breast tumor bearing this amplicon(Siegel, P. et al., Bioessays 22, 554-563 (2000)). The Gab2 gene may beamplified and expressed in breast tumors with the 11q13 amplicon.

Example 9 Overexpression of Gab2 Protein in Breast Cancer Cell Lines andBreast Tumor Cells

[0197] The expression of Gab2 in breast cancer cell lines and breasttumor samples was examined by western blot analysis. The Gab2 proteinwas overexpressed in ˜40% breast cancer cell lines (16 total) examined,such as MDA-MB-134, 468, BT-20, T47D, MDA-MB-435, 21NT. In contrast,Gab2 protein is just above detectable level in immortalized normal humanmammary epithelia cell lines MCF-10A and 184B5. Furthermore, Gab2protein level was high in ˜20% breast tumor samples (total 30 samples)compared to normal breast tissue.

[0198] In collaboration with Dr. Qian Wu at Brown University Hospital inRhode Island, Gab2 expression in five breast tumor samples was examinedby immunohistochemistry using anti-Gab2. Importantly, there was strongGab2 immunostaining in breast carcinoma cells of all the examinedsamples. Strong Gab2 immunostaining was found in carcinoma cells fromboth invasive ductal carcinoma and DCIS tumor samples. In contrast, thenormal mammary epithelial ducts as well as the surrounding normalconnective tissues showed weak Gab2 immunostaining, confirming that Gab2is overexpressed specifically in tumor cells.

Example 10 EGF Induction of Gab2 Tyrosyl Phosphorylation in BreastCancer Cell Line

[0199] Since overexpression of EGFR family members (ErbB2) can promotebreast carcinogenesis, the overexpressed Gab2 may be involved in breastcancer by amplifying EGFR initiated signals. Gab2 tyrosylphosphorylation and its association with signal relay molecules by Gab2immunoprecipitation followed by immunoblotting with p85 and SHP-2antibodies was examined. Gab2 was found to be robustly tyrosylphosphorylated and became associated with p85 and SHP-2 upon EGFstimulation in MDA-MB-486 cells.

Example 11 Generation of Mammary Epithelial Cell Lines OverexpressingGab2

[0200] Gab2 was overexpressed in two well-characterized breastepithelial cell lines to investigate the effects of Gab2 overexpressionon breast cell growth. One breast epithelial cell line was theimmortalized normal epithelial cell line MCF-10A. The other was thebreast cancer cell line MCF-7, which expressed a low level of Gab2.MCF-7 clones that can inducibly express Gab2 using the tetracycline-offexpression system were generated (Gossen, M. & Bujard, H., Proc NatlAcad Sci, USA 89, 5547-5551 (1992)). The advantage of using thisexpression system was that Gab2 expression level can be controlleddepending on the different concentration of tetracycline present in theculture media. These Gab2 overexpressing cell lines will be useful toolsto study the effect of Gab2 on breast cell growth in vitro and tumorformation in mice.

Example 12 Effects of Gab2 Overexpression on Mammary Epithelial CellLines

[0201] Breast cancers mainly arise from breast epithelia, and proceedthrough a series of changes starting with hyperplasia with atypia andprogressing to carcinoma in situ, invasive carcinoma, and eventuallymetastatic disease. At different stages of the carcinogenesis, breastcancer cells become highly proliferative, resistant to apoptosis, growwithout maintaining polarity and/or their dependence on basementmembrane, and more migratory and invasive. Mammary epithelial cell lineswith stable expression of Gab2 were established, whether Gab2overexpression enhances or promotes growth, migration/invasion, andtransformation of mammary epithelial cell lines, such as immortalizedmammary epithelial cells (MCF-10A) and breast cancer cell (MCF-7), invitro can be assessed. The effects of Gab2 overexpression ontumorigenicity in nude mice can also be assessed.

Example 13 Effects of Gab2 Overexpression on the Immortalized MammaryEpithelial Cell Line MCF-10A

[0202] MCF-10A cell growth mainly depends on the presence of EGF in theculture medium since EGF withdrawal causes MCF-10A cells to arrest atG0/G1 (Blagosklonny, M. V. et al., Cancer Res 60, 3425-3428 (2000)) andeventually become apoptotic (Davis, J. W. et al., Carcinogenesis 21,881-886 (2000)). First, the issue of whether Gab2 overexpressionpromotes MCF-10A growth in its growth medium containing EGF can beexamined. An equal number of MCF-10A vector-transfected cells (V) andtwo clones overexpressing Gab2 can be plated in growth media. Cells canbe counted every day for three days by trypan-blue exclusion. If theGab2 overexpressing cells grow faster in this assay, the issue ofwhether Gab2 expression increased MCF-10A proliferation and/or decreasedcell death can be determined. To quantify dead cells, cell samples canbe taken every 24 hours for three days and assayed for apoptotic cellsusing Annexin V (which binds to the cell surface of the early apoptoticcells) reagent.

[0203] The cell cycle length of MCF-10A-V and -Gab2 cell lines can bemeasured to examine whether Gab2 expression increases MCF-10A cellproliferation. Appropriate cells can be pulse labeled with BrdU. Thenevery 3 hours for 24 hours (the typical doubling time of MCF-10A cells),an aliquot of the labeled cells can be taken and the BrdU+ cellsrelative to DNA content can be analyzed by Propidium Iodide (PI).Initially, essentially all BrdU+cells should be in S phase (by PIstaining). Over time, the BrdU+ cells should move progressively from Sphase into G2/M, G1, and back to S phase. The length of each cell cyclephase can be determined by calculating how long it takes BrdU+cells totraverse all the phases. A shorting of either G1, S or G2/M phase ofMCF-10A-Gab2 cells compared to MCF-10A-V cells can indicate which phaseof the cell cycle is affected by the Gab2 overexpression. Since EGF mayinduce Gab2 tyrosine phosphorylation and its association with p85 andSHP-2, but not insulin/IGF-1, Gab2 may promote MCF-10A cell growth(increase cell proliferation and/or survival) through the amplificationof the EGF-initiated signal transduction.

[0204] Two assays can be applied to examine whether Gab2 overexpressioncauses MCF-10A growth independent of polarity and substratum in vitro.First, the ability of MCF-10A-Gab2 cells to undergoanchorage-independent growth can be tested. Equal number of MCF-10A-Vand MCF-10A-Gab2 cells can be seeded in soft agar in growth media. Twoweeks later, the number of colonies containing more than four cells canbe scored as positive (Zelinski, D. P. et al., Cancer Res 61, 2301-2306(2001)). Next, the issued of whether Gab2 overexpression can disrupt theformation of normal mammary tissue structures (acini) by the parentalMCF-10A cells when grown on Matrigel, another property of breast cancercells, can be tested (Weaver, V. M. et al., Semin Cancer Biol 6, 175-184(1995)). Both MCF-10A-V and -Gab2 cells can be seeded on Matrigel coateddishes in growth media. 7-10 days later, the formation of acini (aspherical structure with a single layer of cells surrounding a hollowlumen) can be examined by confocal microscopy. If the formation ofdisorganized cell aggregates on Matrigel and/or the appearance ofpositive colonies in soft agar was observed in cells overexpressingGab2, this could suggest that Gab2 overexpression was oncogenic tonormal breast epithelial cells.

[0205] Another property of aggressive breast cancer cells is that theyare migratory and invasive. The effect of Gab2 expression on migrationand invasion of MCF-10A cells using transwell assay can be examined. Fora migration assay, cells can be seeded in the upper chamber of thetranswell containing semi-permeable filters. A time course (2, 4, 6, 8hours after incubation) of cells migrating to the bottom side of thefilter can be determined. To measure invasion, a similar assay can beperformed except the transwell filter can be coated with Matrigel. Tomeasure the kinetics of cells invading through the matrix and gettingonto the other side of the membrane, a longer time course of measurementcan be followed.

[0206] Because in vitro transformation assays do not always predicttumorigenic potential in vivo, the issue of whether MCF-10A-Gab2 cellscause tumor formation when subcutaneously injected into nude mice can beexamined. If MCF-10A-Gab2 cells are tumorigenic in nude mice, this wouldindicate that Gab2 overexpression may promote tumorigenesis in vivo.

[0207] Since PI-3K and SHP-2 are two major effectors of Gab2 (Gu, H. etal., Mol Cell. 2, 729-740 (1998); Gu, H. et al., Nature 412, 186-190(2001)), the issue of whether Gab2 activated PI-3K and SHP-2 or bothcontribute to the potential increased growth and transformation ofMCF-10A cells can be investigated. To address this question, Gab2mutants that cannot bind PI-3K (ΔPI3K), SHP-2 (ΔSHP2), and both PI-3Kand SHP-2 (ΔPI3K⁺ SHP2) in MCF-10A cells can be expressed using thepBabe-puro retroviral vector. Pools or multiple clones (at least 3 each)of MCF-10A cells expressing similar level of Gab2 wild type (WT) andGab2 mutants can be used for the proliferation, apoptosis,transformation, and migration/invasion assays mentioned above. One ofthe Gab2 ΔPI3K, ΔSHP2 and double mutants would be expected to bedefective in enhancing some of the MCF-10A responses. In case all ofthese Gab2 mutants behave in the same way as Gab2 WT, the effects ofexpressing other Gab2 mutants, such as Gab2 mutant that cannot bind Crk(Crouin, C. et al., FEBS Lett 495, 148-153 (2001)), can be tested onMCF-10A growth. It has been reported that Gab1 binding to Crk correlatesto the ability of Gab 1 to promote transformation by oncogenic Met(Lamorte, L. et al., Oncogene 19, 5973-5981 (2000)).

Example 14 Gab2 Cooperation with ErbB2 on Transformation of MCF-10ACells

[0208] It is conceivable that Gab2 overexpression only enhances MCF-10Aproliferation or survival, but not transformation or tumorigenicity ofMCF-10A due to the limited number of ErbB1 (EGFR) and ErbB2 on thesurface of MCF-10A cells. Overexpression of Gab2 alone may not be enoughto activate oncogenic cascades in MCF-10A cells.

[0209] ErbB2 overexpression in mammary gland can induce breast tumor inmice (Guy, C. T. et al. Proc Natl Acad Sci USA 89, 10578-10582 (1992)).Interestingly, overexpressing ErbB2 cannot transform MCF-10A cells andMCF-10A-ErbB2 cells are not tumorigenic when injected into mice(Giunciuglio, D. et al., Int J Cancer 63, 815-822 (1995)). One possibleexplanation for this is a lack of downstream signaling component forErbB2 oncogenic transformation in MCF-10A cells.

[0210] Therefore, the issue of whether co-overexpression of Gab2 andErbB2 may cause transformation of MCF-10A cells can be addressed. First,MCF-10A cells overexpressing both Gab2 and ErbB2 can be generated.MCF-10A overexpressing ErbB2 (MCF-10A-ErbB2) cells (kindly provided bySam Lee, Beth Israel Deaconess Medical Center) can be infected withpBabe-puro virus alone, or with pBabe-puro virus containing Gab2 WT, orwith pBabe-puro virus containing different Gab2 mutants such as ΔPI3Kand ΔSHP2. MCF-10A-ErbB2 clones overexpressing a similar level of Gab2WT and mutants can be screened by immunoblotting with Gab2 antibodies.At least two different clones expressing each Gab2 protein can be usedfor the subsequent studies. Next, the issue of whetherMCF-10A-ErbB2-Gab2 cells become transformed can be examined by assayingtheir ability to grow in soft agar, form disorganized aggregates onMatrigel (inability to form acini), and induce tumor formation wheninjected into nude mice. If MCF-10A-ErbB2-Gab2 cells display all of thetransforming phenotypes, this would suggest that overexpression of Gab2together with ErbB2 overexpression can contribute to breast tumorformation in vivo.

[0211] To examine how Gab2 may cooperate with ErbB2 to cause thetransformation of MCF-10A cells, one can test whether MCF-10A-ErbB2clones expressing different Gab2 mutants (as discussed) can grow in softagar, disrupt acini formation on Matrigel, and/or form tumor in mice. Itwould be expected that the Gab2-API3K mutant may be defective in thesetransformation assays, suggesting that Gab2 activation of PI-3K wasimportant for breast carcinogenesis, consistent with the known role ofPI-3K in oncogenic transformation (Cantley, L. & Neel, B., Proc NatlAcad Sci USA 96, p4240-4245 (1999)). However, it cannot be ruled outthat the Gab2-DSHP2 may be also defective in some of the transformationassays, considering that SHP-2 mainly functions as a positive signaltransducer downstream of RTK including EGFR (Chen, B. et al., Nat Genet24, 296-299 (2000)).

Example 15 Effects of Overexpressing Gab2 on the Growth/Tumorigenicityof Breast Cancer Cell MCF-7

[0212] Activation or inactivation of additional key molecules besideErbB2 and Gab2 may be required for MCF-10A cells to become tumorigenicin mice. If overexpression of Gab2 and ErbB2 only causes transformationof MCF-10A cells in vitro (i.e., anchorage independent growth), but nottumorigenicity in mice, this would suggest that Gab2 may not be the onlycritical downstream target mediating the ErbB2 oncogenic signal.

[0213] To see whether overexpression of Gab2 can promote tumorformation, one can test whether overexpression of Gab2 will enhance thegrowth/or tumorigenicity of an already carcinogenic but relativelynon-aggressive breast cancer cell MCF-7. MCF-7 cells express detectablelevel of ErbB2 (Cuello, M. et al, Cancer Res 61, 4892-4900 (2001)) andGab2. It has lower invasive property (Johnson, M. D. et al., Cancer Res53, 873-877 (1993)) and forms tumors in nude mice with longer latency(Yue, W. & Brodie, A., J Steroid Biochem Mol Biol 44, 671-673 (1993)). AMCF-7 cell line that expresses Gab2 inducibly using the tetracycline(Tet)-off system has been generated. The Tet-off system has been usedwidely to achieve inducible expression of a gene of interest in culturedcells (Gossen, M. & Bujard, H., Proc Natl Acad Sci, USA 89, 5547-5551(1992)) as well as in mice (Huettner, C. S. et al., Nat. Genet. 24,57-60 (2000)) upon decreasing or withdrawing tetracycline (Tet) fromculture media and drinking water.

[0214] First, the issue of whether inducing the expression of Gab2 willenhance transforming properties of MCF-7 cells in vitro can be examined.For example, soft agar and transwell invasion assay using MCF-7 Gab2cells in the absence or presence of Tet can be performed. Ifoverexpression of Gab2 enhances MCF-7 transformation, one would expectthat the formation of larger colonies in the absence of tetracycline(Gab2 is overexpressed) compared to in the presence of tetracycline,and/or that MCF-7 cells will invade through the Matrigel faster in theabsence of Tet. Next, the tumorigenic property of MCF-7-Gab2 cells canbe tested by injecting the cells into nude mice that have beenmaintained on Tet-containing water for several days. After injection,half of the injected mice can be still kept on Tet containing water andthe other half of the mice can be on Tet free water. Typically, parentalMCF-7 cells form visible tumors in nude mice about two months afterinoculation. If tumors are observed earlier in mice on Tet free water,this would suggest that overexpression of Gab2 promotes enhanced breasttumor formation. To confirm that the tumors with early onset are due toGab2 overexpression, Gab2 expression in these tumors can be examined byimmunoblotting with Gab2 antibodies.

Example 16 To Investigate whether Gab2 is Necessary and Sufficient toPromote Breast Cancer Alone or in Cooperation with the MMTV-NeuTransgene

[0215] Although studies from Gab2 overexpression in breast epithelialcell lines in vitro suggest a role of Gab2 in breast carcinogenesis, theeffect of Gab2 overexpression can be varied depending on different celllines used in the studies. To test whether Gab2 expression is requiredfor breast tumor formation and progress, mouse genetic approaches can beused.

Example 17 Overexpression of Gab2 in a Mammary Gland Using TransgenicApproach

[0216] The issue of whether Gab2 overexpression is sufficient to causebreast tumor in mice can be investigated. To address this question,transgenic mice overexpressing Gab2 specifically in the mammary glandcan be generated. The transgenic construct, in which the Gab2 cDNA hastwo HA tags at its C-terminus, can be placed under the control of a WAPpromoter, which is only active in mammary glands, to generate WAP-Gab2transgenic mice. The generation of WAP-Gab2 transgenic mice in FvBstrain lines can take about 6-8 months, during which western blotanalysis can be used to check whether the founder transgenic linesspecifically overexpress Gab2 in the mammary gland. Gab2 proteinexpressed from the transgene should run slower in SDS-PAGE because ofthe HA tags. By ten months, a large number of WAP-Gab2 transgenic micethat can be used to set up big breeding colonies for tumorigenic studiescan be generated.

[0217] To assess whether Gab2 overexpression in a mammary gland issufficient to cause a breast tumor, a large breeding colony can beestablished by mating the WAP-Gab2 transgenic mice with the wild typeFvB mice. Since it is hard predict when breast tumor will form inWAP-Gab2 mice, these mice can be kept for the duration of their normallifespan.

[0218] Since Gab2 may cooperate with ErbB2, the issue of whether Gab2overexpression shortens the latency of breast tumor formation inMMTV-neu transgenic mice can be studied, in case WAP-Gab2 mice alone donot develop breast tumors. Therefore, a WAP-Gab2/MMTV-neu cross can besetup at the same time as the setup of the WAP-Gab2 mice interbreeding.Since MMTV-neu mice (obtained from Jackson ImmunoResearch Laboratory,West Grove, Pa.) typically develop mammary tumors around 6-7 month(Muller, W. J. et al., Cell 54, 105-115 (1988)), the question of whetherGab2 overexpression in a mammary gland potentiates breast tumorformation induced by MMTV-neu transgene can be answered.

[0219] One possible outcome of this approach is that a WAP-Gab2transgene alone may not cause a breast tumor, but it may potentiatebreast tumor formation-induced MMTV-neu (i.e., it may significantlyshorten the latency of the breast tumor onset). This would stronglyimply that Gab2 overexpression in tumors is actively involved in thebreast tumor progression by cooperation with one or more otheroncogenes.

Example 18 Analyzing a Cross Between Gab2 Knockout Mice and MMTV-neuTransgenic Mice

[0220] Since published biochemical data suggest that Gab2 functionsdownstream of EGFR (ErbB1), the issue of whether Gab2 is required formammary tumor induction in MMTV-neu mice can be investigated. To addressthis question, MMTV-neu mice can be crossed with the Gab2 knockout (−/−)mice. Gab2−/− mice are healthy, except that they have impaired allergyresponse. As a control, Gab2−/− mice can be crossed with MMTV-myc mice(developing mammary tumor about 8-10 months) since MMTV-neu and MMTV-mycinduce mammary tumors through a different mechanism. MMTV-neu inductionof breast tumors requires cyclin D1, MMTV-myc does not (Yu, Q. et al.,Nature 411, 1017-1021 (2001)).

[0221] The Gab2−/− mice can be crossed with either MMTV-neu or myc mice.Gab2+/−:MMTV-neu and Gab2+/−:MMTV-myc mice will result from theseinitial crosses (takes about three months). Gab2+/−:MMTV-neu mice can beinterbred to generate Gab2−/− :MMTV-neu and Gab2+/+:MMTV-neu mice, andGab2+/−:MMTV-myc mice can be interbred to generate Gab2−/−:MMTV-myc andGab2+/+:MMTV-myc mice. The progeny can be monitored for breast tumorformation in the progeny over a 14-month period, which should allowGab2−/−:MMTV-neu or Gab2−/−:MMTV-myc mice to show delayed onset ofbreast tumor formation.

[0222] If Gab2−/−:MMTV-neu mice show no or decreased breast tumordevelopment within fourteen months, and Gab2−/−:MMTV-myc mice developtumors with the same kinetics as Gab2+/+:MMTV-myc mice, this wouldsuggest the genetic model that MMTV-Neu cause breast cancer via aGab2->cyclin D1 cascade. It is also possible that loss of Gab2 has noeffects on the latency of breast tumor development in either MMTV-neu orMMTV-myc mice. This would suggest that breast tumor induction by theMMTV-neu or myc does not require normal endogenous levels of Gab2.

[0223] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

1 37 1 6 PRT Unknown Murine 1 Lys Ala Lys Pro Thr Pro 1 5 2 6 PRTUnknown Murine 2 Thr Val Ile Asp Glu Leu 1 5 3 21 DNA ArtificialSequence primer sequence 3 ccttgacctg agaaacaaca c 21 4 6 PRT UnknownMurine 4 Leu Asp Leu Arg Asn Asn 1 5 5 666 PRT Unknown Murine 5 Met SerGly Gly Gly Gly Asp Asp Val Val Cys Thr Gly Trp Leu Arg 1 5 10 15 LysSer Pro Pro Glu Lys Lys Leu Arg Arg Tyr Ala Trp Lys Lys Arg 20 25 30 TrpPhe Ile Leu Arg Ser Gly Arg Met Ser Gly Asp Pro Asp Val Leu 35 40 45 GluTyr Tyr Lys Asn Glu His Ser Lys Lys Pro Leu Arg Ile Ile Asn 50 55 60 LeuAsn Leu Cys Glu Gln Val Asp Ala Gly Leu Thr Phe Asn Lys Lys 65 70 75 80Glu Leu Gln Asp Ser Phe Val Phe Asp Ile Lys Thr Ser Glu Arg Thr 85 90 95Phe Tyr Leu Val Ala Glu Thr Glu Ala Asp Met Asn Lys Trp Val Gln 100 105110 Ser Ile Cys Gln Ile Cys Gly Phe Asn Gln Ala Glu Glu Ser Thr Asp 115120 125 Ser Leu Arg Asn Leu Ser Ser Ala Ser His Gly Pro Arg Ser Ser Pro130 135 140 Ala Glu Phe Ser Ser Ser Gln His Leu Leu Arg Glu Arg Lys SerSer 145 150 155 160 Ala Pro Ser His Ser Ser Gln Pro Thr Leu Phe Thr PheGlu Pro Pro 165 170 175 Val Ser Ser His Met Gln Pro Thr Leu Ser Thr SerAla Pro Gln Glu 180 185 190 Tyr Leu Tyr Leu His Gln Cys Ile Ser Arg ArgThr Glu Asn Ala Arg 195 200 205 Ser Ala Ser Phe Ser Gln Gly Thr Arg GlnLys Ser Asp Thr Ala Val 210 215 220 Gln Lys Leu Ala Gln Ser Asn Gly HisCys Ile Asn Gly Val Gly Gly 225 230 235 240 Gln Val His Gly Phe Tyr SerLeu Pro Lys Pro Ser Arg His Asn Thr 245 250 255 Glu Phe Lys Asp Ser ThrTyr Asp Leu Pro Arg Ser Leu Ala Ser His 260 265 270 Gly His Thr Lys SerSer Leu Thr Gly Ser Glu Thr Asp Asn Glu Asp 275 280 285 Val Tyr Thr PheLys Met Pro Ser Asn Thr Leu Cys Arg Glu Leu Gly 290 295 300 Asp Leu LeuVal Asp Asn Met Asp Val Pro Thr Thr Pro Leu Ser Ala 305 310 315 320 TyrGln Ile Pro Arg Thr Phe Thr Leu Asp Lys Asn His Asn Ala Met 325 330 335Thr Val Ala Thr Pro Gly Asp Ser Ala Ile Ala Pro Pro Pro Arg Pro 340 345350 Pro Lys Pro Ser Gln Ala Glu Thr Ser Gln Trp Gly Ser Ile Gln Gln 355360 365 Arg Pro Pro Ile Ser Glu Asn Ser Arg Ser Val Ala Ala Thr Ile Pro370 375 380 Arg Arg Asn Thr Leu Pro Ala Met Asp Asn Ser Arg Leu His ArgAla 385 390 395 400 Ser Ser Cys Glu Thr Tyr Glu Tyr Pro Ala Arg Gly SerGly Glu Ser 405 410 415 Ala Ser Trp Ser Ala Glu Pro Pro Gly Lys Thr AlaVal Gly Arg Ser 420 425 430 Asn Ser Ala Ser Ser Asp Asp Asn Tyr Val ProMet Asn Pro Gly Ser 435 440 445 Ser Thr Leu Leu Ala Met Glu Arg Pro GlyAsp Asn Ser Gln Ser Val 450 455 460 Tyr Ile Pro Met Ser Pro Gly Pro HisHis Phe Asp Pro Leu Gly Tyr 465 470 475 480 Pro Ser Thr Ala Leu Pro IleHis Arg Gly Pro Ser Arg Gly Ser Glu 485 490 495 Ile Gln Pro Pro Pro ValAsn Arg Asn Leu Lys Pro Asp Arg Lys Ala 500 505 510 Lys Pro Thr Pro LeuAsp Leu Arg Asn Asn Thr Val Ile Asp Glu Leu 515 520 525 Pro Phe Lys SerPro Val Thr Lys Ser Trp Ser Arg Ile Asn Ser His 530 535 540 Thr Phe AsnSer Ser Ser Ser Gln Tyr Cys Arg Pro Ile Ser Thr Gln 545 550 555 560 SerIle Thr Ser Thr Asp Ser Gly Asp Ser Glu Glu Asn Tyr Val Pro 565 570 575Met Gln Asn Pro Val Ser Ala Ser Pro Val Pro Ser Gly Thr Asn Ser 580 585590 Pro Ala Pro Lys Lys Ser Thr Gly Ser Val Asp Tyr Leu Ala Leu Asp 595600 605 Phe Gln Pro Gly Ser Pro Ser Pro His Arg Lys Pro Ser Thr Ser Ser610 615 620 Val Thr Ser Asp Glu Lys Val Asp Tyr Val Gln Val Asp Lys GluLys 625 630 635 640 Thr Gln Ala Leu Gln Asn Thr Met Gln Glu Trp Thr AspVal Arg Gln 645 650 655 Ser Ser Glu Pro Ser Lys Gly Ala Lys Leu 660 6656 1965 DNA Unknown Murine 6 atgagcggcg gcggcggcga cgacgtggtg tgtaccggctggctgaggaa atcgcctccc 60 gagaagaagt tgaggcgcta tgcctggaag aaacgctggtttatacttcg gagtggccga 120 atgagtggag atccagatgt tctggaatac tacaagaatgagcactccaa gaaacccctg 180 cggatcatca acctgaactt gtgtgagcag gtggatgcaggcctgacctt caacaagaaa 240 gagctgcagg atagttttgt gtttgatatc aagaccagcgagcgcacatt ttacctggtg 300 gctgagacag aggctgacat gaataagtgg gtccagagcatctgccagat ctgcggcttc 360 aatcaggctg aagagagcac agactccctg aggaacctttcttcagccag tcatggtccc 420 cgctcttctc cagctgagtt cagctccagt cagcacctgctccgagaacg gaagtcctca 480 gcccttcaca ctctagccag cctactttat tcacgtttgagccccctgtg tcaagccaca 540 tgcagcctac ctgtccacca gtgcacctca ggagtatctctacttgcacc agtgcataag 600 cagaaggaca gaaaatgcaa ggagtgccag cttctctcagggcacccggc agaagagtga 660 tacagctgtg caaaaacttg cccagagcaa tggacactgtatcaacggcg tcggaggtca 720 agtccatggc ttctatagcc ttcccaagcc aagccgacacaatacagaat tcaaagacag 780 tacttatgat ctcccacgga gcctggcttc ccatggccacaccaagagca gcctcacagg 840 gtctgagact gataacgagg atgtgtacac cttcaagatgcccagcaaca ccctgtgtcg 900 ggaacttgga gacctccttg tggacaatat ggatgtcccaaccactcctc tctcagccta 960 ccagatccct agaacattca cactggacaa gaaccacaatgccatgacag tggccactcc 1020 tggagattca gccatagctc ccccaccccg gccacccaagccaagtcagg cagaaacatc 1080 tcaatggggc agcattcagc aaagacctcc aatcagcgaaaatagcagat ctgtagctgc 1140 tactatcccc aggcgcaata ccctccctgc aatggacaacagccgactcc atcgagcttc 1200 ttcctgtgag acctacgagt acccggcacg aggcagtggggaaagtgcca gctggtctgc 1260 tgaacctcca ggaaagactg ccgtaggtcg atcaaatagtgccagctctg atgacaacta 1320 cgtgcccatg aacccaggtt cttctaccct gctggctatggaacgaccag gggacaactc 1380 ccagagtgtc tacatcccca tgagcccagg accccatcactttgacccac ttggctaccc 1440 gccacagccc ttcctattca cagaggcccc agccgaggaagtgagatcca gccacccccg 1500 gtcaaccgaa acctcaagcc tgacagaaaa gcaaagccaacaccccttga cctgagaaac 1560 aacactgtca tcgatgacct gcccttcaag tcacctgtcaccaagtcttg gtccaggatc 1620 aacagccaca cctttaactc cagttcctcc cagtactgccgtcactatgt ccctatgcaa 1680 aacccagtat ctgcatcccc tgttcccagt ggcactaacagcccagctcc aaagaagagt 1740 actggcagtg tggattatct cgccctggac ttccagccgggctccccaag ccctcaccgc 1800 aagccatcca catcatctgt cacatcagat gagaaggtagactatgtcca agtggataaa 1860 gagaagaccc aggccctgca gaacaccatg caggagtggacagatgtgcg ggcagtcctc 1920 cgaaccttcc aagggtgcca agctgtaatg aagggggccaccaag 1965 7 12 PRT Unknown Murine 7 Gln Leu Glu Glu Asp Tyr Tyr Gly LeuSer Lys Gly 1 5 10 8 7 PRT Unknown Murine 8 Thr Gln Ala Leu Gln Asn Thr1 5 9 11 PRT Unknown Murine 9 Asp Ser Thr Tyr Asp Leu Pro Arg Ser LeuAla 1 5 10 10 9 PRT Unknown Murine 10 Pro Ser Arg His Asn Thr Glu PheLys 1 5 11 5 PRT Unknown Murine 11 Val Gln Val Asp Lys 1 5 12 11 PRTUnknown Murine 12 Glu Leu Gln Asp Ser Phe Val Phe Asp Ile Lys 1 5 10 1317 PRT Unknown Murine 13 Ala Lys Pro Thr Pro Leu Asp Leu Arg Asn Asn ThrVal Ile Asp Glu 1 5 10 15 Leu 14 17 PRT Unknown Murine 14 Ser Ser LeuThr Gly Ser Glu Thr Asp Asn Glu Asp Val Tyr Thr Phe 1 5 10 15 Lys 15 4PRT Unknown Murine 15 Tyr Lys Asn Glu 1 16 4 PRT Unknown Murine 16 TyrSer Leu Pro 1 17 4 PRT Unknown Murine 17 Tyr Asp Leu Pro 1 18 4 PRTUnknown Murine 18 Tyr Gln Ile Pro 1 19 4 PRT Unknown Murine 19 Tyr GluTyr Pro 1 20 4 PRT Unknown Murine 20 Tyr Val Pro Met 1 21 4 PRT UnknownMurine 21 Tyr Ile Pro Met 1 22 4 PRT Unknown Murine 22 Tyr Val Pro Met 123 4 PRT Unknown Murine 23 Tyr Leu Ala Leu 1 24 4 PRT Unknown Murine 24Tyr Val Gln Val 1 25 4 PRT Unknown Murine 25 Lys Ser Pro Pro 1 26 118PRT Unknown Murine 26 Met Ser Gly Gly Gly Gly Asp Asp Val Val Cys ThrGly Trp Leu Arg 1 5 10 15 Lys Ser Pro Pro Glu Lys Lys Leu Arg Arg TyrAla Trp Lys Lys Arg 20 25 30 Trp Phe Ile Leu Arg Ser Gly Arg Met Ser GlyAsp Pro Asp Val Leu 35 40 45 Glu Tyr Tyr Lys Asn Glu His Ser Lys Lys ProLeu Arg Ile Ile Asn 50 55 60 Leu Asn Leu Cys Glu Gln Val Asp Ala Gly LeuThr Phe Asn Lys Lys 65 70 75 80 Glu Leu Gln Asp Ser Phe Val Phe Asp IleLys Thr Ser Glu Arg Thr 85 90 95 Phe Tyr Leu Val Ala Glu Thr Glu Ala AspMet Asn Lys Trp Val Gln 100 105 110 Ser Ile Cys Gln Ile Cys 115 27 115PRT Unknown Murine 27 Met Ser Gly Gly Glu Val Val Cys Ser Gly Trp LeuArg Lys Ser Pro 1 5 10 15 Pro Glu Lys Lys Leu Lys Arg Tyr Ala Trp LysLys Arg Trp Phe Val 20 25 30 Leu Arg Ser Gly Arg Leu Thr Gly Asp Pro AspVal Leu Glu Tyr Tyr 35 40 45 Lys Asn Asp His Ala Lys Lys Pro Ile Arg IleIle Asp Leu Asn Leu 50 55 60 Cys Gln Gln Val Asp Ala Gly Leu Thr Phe AsnLys Lys Glu Phe Glu 65 70 75 80 Asn Ser Tyr Ile Phe Asp Ile Asn Thr IleAsp Arg Ile Phe Tyr Leu 85 90 95 Val Ala Asp Ser Glu Glu Glu Met Asn LysTrp Val Arg Cys Ile Cys 100 105 110 Asp Ile Cys 115 28 112 PRT UnknownMurine 28 Met Asp Arg Thr Phe Tyr Glu Gly Trp Leu Ile Lys Ser Pro ProThr 1 5 10 15 Lys Arg Ile Trp Arg Ala Arg Trp Arg Arg Arg Tyr Phe ThrLeu Lys 20 25 30 Gln Gly Glu Ile Pro Glu Gln Phe Cys Leu Glu Tyr Tyr ThrAsp His 35 40 45 Asn Cys Arg Lys Leu Lys Gly Val Ile Asp Leu Asp Gln CysGlu Gln 50 55 60 Val Asp Cys Gly Leu Arg Leu Glu Asn Arg Lys Gln Lys PheGln Tyr 65 70 75 80 Met Phe Asp Ile Lys Thr Pro Lys Arg Thr Tyr Tyr LeuAla Ala Glu 85 90 95 Thr Glu Ala Asp Met Arg Asp Trp Val Asn Cys Ile CysGln Val Cys 100 105 110 29 4 PRT Unknown Murine 29 Leu Glu Tyr Tyr 1 3083 PRT Unknown Murine 30 Pro Met Asn Pro Asn Ser Pro Pro Arg Gln His SerSer Ser Phe Thr 1 5 10 15 Glu Pro Ile Gln Glu Ala Asn Tyr Val Pro MetThr Pro Gly Thr Phe 20 25 30 Asp Phe Ser Ser Phe Gly Met Gln Val Pro ProPro Ala His Met Gly 35 40 45 Phe Arg Ser Ser Pro Lys Thr Pro Pro Arg ArgPro Val Pro Val Ala 50 55 60 Gln Cys Glu Pro Pro Pro Val Asp Arg Asn LeuLys Pro Asp Arg Lys 65 70 75 80 Val Lys Pro 31 4 PRT Unknown Murine 31Pro Met Asn Pro 1 32 4 PRT Unknown Murine 32 Pro Pro Pro Val 1 33 8 PRTUnknown Murine 33 Arg Asn Leu Lys Pro Asp Arg Lys 1 5 34 72 PRT UnknownMurine 34 Pro Met Asn Pro Gly Ser Ser Thr Leu Leu Ala Met Glu Arg ProGly 1 5 10 15 Asp Asn Ser Gln Ser Val Tyr Ile Pro Met Ser Pro Gly ProHis His 20 25 30 Phe Asp Pro Leu Gly Tyr Pro Ser Thr Ala Leu Pro Ile HisArg Gly 35 40 45 Pro Ser Arg Gly Ser Glu Ile Gln Pro Pro Pro Val Asn ArgAsn Leu 50 55 60 Lys Pro Asp Arg Lys Ala Lys Pro 65 70 35 4 PRT UnknownMurine 35 Tyr Cys Arg Pro 1 36 4 PRT Unknown Murine 36 Tyr Leu Tyr Leu 137 9 PRT Unknown VARIANT 1 Xaa = Gln or Glu or Pro 37 Xaa Xaa Xaa XaaXaa Thr Glu Phe Lys 1 5

What is claimed is:
 1. An isolated nucleic acid molecule comprising: a.Gab2 or a fragment, derivative or mutation thereof; b. the nucleic acidsequence of SEQ ID NO. 6; c. a sequence which encodes a polypeptidecomprising amino acid sequence of SEQ IDNO. 5; d. a nucleic acidsequence with 90% sequence identity to SEQ ID NO. 6; e. a complementarystrand of the sequence of (b), (c) or (d); f. RNA sequences transcribedfrom sequences (b), (c), (d) or (e), or a fragment or mutation thereof;g. DNA sequences that hybridize to the sequence of (b), (d) or (e). 2.An expression vector comprising the nucleic acid molecule according toclaim 1, or the nucleic acid molecule of claim 1 where the molecule hasbeen mutated.
 3. A host cell comprising the expression vector of claim2.
 4. The protein either encoded by the nucleic acid sequence of SEQ IDNO: 6 or comprising the amino acid sequence of SEQ ID NO:
 5. 5. Anantibody specific to the protein of claim
 4. 6. A vector comprising thenucleic acid molecule of claim
 1. 7. A probe comprising the nucleic acidmolecule of claim
 1. 8. A transgenic non-human mammal with a genomecomprising a disruption of the Gab2 gene such that the mammal lacks orhas reduced levels of functional Gab2 protein, and wherein the mammalexhibits an altered responsiveness to cytokine, growth factor, hormoneor antigen stimulation.
 9. A transgenic non-human mammal with a genomecomprising an alteration of the Gab2 gene such that the mammal hasincreased levels of functional Gab2 protein, and wherein the mammalexhibits an altered responsiveness to cytokine, growth factor, hormoneor antigen stimulation.
 10. The transgenic non-human mammal of claim 8or claim 9 wherein the mammal is a transgenic mouse.
 11. The transgenicmouse of claim 8, claim 9 or claim 10 wherein the genome comprises adisruption of the Gab2 gene selected from the group consisting of ahomozygous disruption and a heterozygous disruption.
 12. Use of an agentwhich inhibits a Gab2 interaction with an associated protein for themanufacture of a medicament for preventing or treating a GAb2-mediatedinjury.
 13. The use of claim 12 wherein the Gab2 interaction with anassociated protein is in response to an extracellular stimulation. 14.The method of claim 13 wherein the extracellular stimulus is a cytokine,growth factor, hormone or antigen.
 15. The use of claim 12, claim 13 orclaim 14 wherein the Gab2-mediated injury is an allergic response, aneoplastic disease, or an immune disorder.
 16. The use of any one ofclaims 12 to 15 wherein the agent is selected from the group consistingof proteins, polypeptides, antibodies, oligonucleotides, smallmolecules, natural product inhibitors, mutants of Gab2, and mutants ofGab2-associated molecules.
 17. The use of any one of claims 12 to 15wherein the agent is an oligonucleotide antisense to the nucleic acidsequence of Gab2, or antisense to a Gab2 homolog, fragment,complementary sequence, or mutant.
 18. The use of any one of claims 12to 15 wherein the agent is a mutant Gab2, or fragment thereof, whichcompetes with Gab2 for interaction with its associated proteins.
 19. Theuse of any one of claims 12 to 18 wherein tyrosyl phosphorylation ofGab2 is prevented by administration of the agent.
 20. The use of any oneof claims 12 to 19 wherein expression of Gab2 is inhibited or eliminatedby administration of the agent.
 21. The use of any one of claims 12 to20 wherein the agent is administered for nasal, topical or systemic use.22. The use of any one of claims 12 to 20 wherein the agent is anoligonucleotide administered as an insert in a gene therapy vector. 23.The use of any one of claims 12 to 22 wherein the associated protein isselected from the group consisting of p85, PI-3K, a protein containing aSH-2 domain, a protein containing a SH-3 domain, a protein containing aPH domain and a protein containing a WW domain.
 24. The use of any oneof claims 12 to 21 or claim 23 wherein the agent inhibits the responseof mast cells to FceRI receptor stimulation by administration to themast cells.
 25. The use of any one of claims 12 to 21 or claim 23wherein the Gab2-mediated injury is an allergic response and the agentinhibits said Gab2 interaction in response to an extracellular stimulus,thereby preventing activation of a Gab2-mediated signaling cascade. 26.The use of claim 24 or claim 25 wherein the response is degranulation,cytokine gene expression or anaphylaxis.
 27. The use of any one ofclaims 12 to 20, claim 22 or claim 23 wherein the Gab2-mediated injuryis a neoplastic disease and the agent prevents activation of aGab2-mediated signaling cascade.
 28. The use of claim 27 wherein theneoplastic disease is selected from the group consisting of leukemia,prostate cancer and breast cancer.
 29. The use of any one of claims 12to 20, claim 22 or claim 23 wherein the Gab2-mediated injury is breastcancer and the agent prevents activation of a Gab2-mediated signalingcascade.
 30. The use of any one of claims 12 to 15 or claims 19 to 29wherein the agent is a short, double-stranded RNA molecule or a short,double-stranded RNA/DNA combination, directed against Gab2 nucleic acidsequence for the purpose of decreasing or eliminating Gab2 geneexpression.
 31. A method of detecting upregulation of Gab2 product in apatient with a neoplastic disorder comprising testing a sample from apatient suspected of having a neoplastic disorder with the probe ofclaim
 7. 32. A method of identifying a drug to be administered to treata Gab2-mediated condition in a mammal in which the condition occurs,comprising: (a) producing a mouse that is a model of the condition; (b)administering to the mouse a drug to be assessed for its effectivenessin treating or preventing the condition; and (c) assessing the abilityof the drug to treat or prevent the condition, wherein if the drugreduces the extent to which the condition is present or progresses, thedrug is a drug to be administered to treat the condition.
 33. The methodof claim 32 wherein the mouse contains a genetic mutation causing aGab2-mediated condition.
 34. The method of claim 32 or claim 33 whereinthe Gab2-mediated condition is an allergic response, a neoplasticdisease, or an immune disorder.
 35. Isolated RNA that mediates RNAinterference of Gab2 mRNA.
 36. Isolated RNA of claim 35 that comprises aterminal 3′ hydroxyl group.
 37. Isolated RNA of claim 35 or claim 36which is chemically synthesized RNA or an analog of a naturallyoccurring RNA.
 38. An analog of isolated RNA of claim 35, claim 36 orclaim 37 wherein the analog differs from the RNA of Gab2 by theaddition, deletion, substitution or alteration of one or morenucleotides.
 39. Isolated RNA that inactivates the Gab2 gene bytranscriptional silencing.
 40. A method of mediating RNA interference ofmRNA of the Gab2 gene in a cell or organism comprising: a. introducingRNA of sufficient length which targets the mRNA of the Gab2 gene fordegradation into the cell or organism; and b. maintaining the cell ororganism produced in (a) under conditions under which degradation of themRNA occurs, thereby mediating RNA interference of the mRNA of the genein the cell or organism.
 41. The method of claim 40 wherein the RNA of(a) is a chemically synthesized RNA or an analog of naturally occurringRNA.
 42. A method of mediating RNA interference of mRNA of the Gab2 genein a cell or organism in which RNA interference occurs, comprising: a.combining double-stranded RNA that corresponds to a sequence of the Gab2gene with a soluble extract that mediates RNA interference, therebyproducing a combination; b. maintaining the combination produced in (a)under conditions under which the double-stranded RNA is processed to RNAof sufficient length, thereby producing processed RNA of sufficientlength; c. isolating the RNA of sufficient length produced in (b); d.introducing RNA isolated in (c) into the cell or organism; and e.maintaining the cell or organism produced in (d) under conditions underwhich degradation of mRNA of the Gab2 gene occurs, thereby mediating RNAinterference of the mRNA of the Gab2 gene in the cell or organism. 43.The method of claim 42 wherein the processed RNA of step (b) is of fromabout 21 to 23 nucleotides.
 44. A method of mediating RNA interferenceof mRNA of the Gab2 gene in a cell or organism in which RNA interferenceoccurs, comprising: (a) introducing into the cell or organism RNA ofsufficient length that mediates RNA interference of mRNA of the Gab2gene, thereby producing a cell or organism that contains the RNA; and(b) maintaining the cell or organism that contains the RNA underconditions under which RNA interference occurs, thereby mediating RNAinterference of mRNA of the Gab2 gene in the cell or organism.
 45. Theuse of an agent for the manufacture of a medicament for treating adisease or condition associated with the presence of a Gab2 protein inan individual wherein the agent comprises RNA of sufficient length thattargets the mRNA of the Gab2 gene for degradation.
 46. The use of claim45 wherein RNA of sufficient length is chemically synthesized or ananalog of RNA that mediates RNA interference.
 47. The use of claim 45 orclaim 46 wherein the agent is used in a method according to any one ofclaims 40 to
 44. 48. The use of any one of claims 45 to 47 wherein theagent comprises the indicated RNA of any one of claims 35 to 39.